Holt was sworn in as prime minister on 26 January 1966, following the retirement of Robert Menzies six days earlier. He won the leadership election unopposed, with William McMahon elected as his deputy. His swearing in was delayed by the death of Defence Minister Shane Paltridge; he and Menzies both served as pallbearers at Paltridge's state funeral on 25 January. Holt was the first Australian prime minister born in the 20th century and the first born after federation. He was almost fourteen years younger than his predecessor, but, at the age of 57, was still the fourth-oldest man to assume the office. He had been an MP for over 30 years before becoming prime minister, still the longest wait for any non-caretaker Prime Minister. The only person who had a longer wait was his caretaker successor John McEwen, who had served 33 years before ascending to the post. Stylistically, Holt was more informal and contemporary than Menzies, and his wife accompanied him into the political spotlight. He gave the media an unprecedented level of access, and was the first prime minister to conduct regular press conferences and grant regular television interviews. His press secretary, Tony Eggleton, accompanied him virtually every time he travelled.
Holt's initial cabinet was virtually unchanged from that of his predecessor. John Gorton and Les Bury were promoted to replace Menzies and Paltridge, but there were no other changes in composition. There were also no major changes in portfolio, outside of McMahon's promotion to Treasurer in place of Holt. A notable addition to the outer ministry was Senator Annabelle Rankin as Minister for Housing – the first woman to hold a ministerial portfolio. A minor reshuffle occurred after the 1966 election, with Doug Anthony and Ian Sinclair added to cabinet and Charles Barnes demoted to the outer ministry. The only new government department created during Holt's tenure was the Department of Education and Science, established in December 1966, which was the first federal department specific to either of those areas.
On 26 November 1966, Holt fought his first and only general election as prime minister, winning a somewhat unexpected landslide victory. The Coalition secured 56.9 percent of the two-party-preferred vote, gaining 10 seats and bringing its total number of seats in the House of Representatives to 82 out of 124; the Liberal Party was only two seats away from forming majority government in its own right. It was a higher margin victory of victory than Menzies had achieved in eight elections as Liberal leader, and was the Labor Party's worst electoral defeat in 31 years.
As prime minister, Holt continued the liberalisation of immigration law that he had begun as Minister for Immigration. When he came to office, what remained of the White Australia policy was upheld by ministerial decree rather than by explicit legislation. In March 1966, the residency requirement for naturalisation was changed to a uniform five years; it had previously been 15 years for non-whites. Discriminatory provisions relating to family reunification were also removed. As a result, in the two years after March 1966 around 3,000 Asian immigrants were granted Australian citizenship, compared with 4,100 in the preceding two decades. Additionally, Immigration Minister Hubert Opperman announced that potential immigrants to Australia would be assessed solely "on the basis of their suitability as settlers, their ability to integrate readily, and their possession of qualifications which are in fact positively useful to Australia"; non-whites had previously had to demonstrate that they were "highly qualified and distinguished" to gain entry.
Personal diplomacy was Holt's strong point – he believed diplomatic ties could be strengthened by making intimate connections with other world leaders. This approach was disliked by his external affairs minister, Paul Hasluck, who in his memoirs accused him of believing in "instant diplomacy" and crediting his personal charms for advances made by diplomatic officials. As prime minister, Holt's first overseas trip was to South-East Asia in April 1966, where he visited Malaysia, Singapore, South Vietnam, and Thailand. He toured Cambodia, Laos, South Korea, and Taiwan in March and April 1967, and had planned to visit Burma, India, Indonesia, Japan, and Pakistan in 1968. Most of those countries had never before been visited by an Australian prime minister. There were also a number of reciprocal visits from East Asian leaders, including Eisaku Satō of Japan, Souvanna Phouma of Laos, and Thanom Kittikachorn of Thailand. The most controversial of those occurred in January 1967, when Prime Minister Nguyễn Cao Kỳ of South Vietnam visited on Holt's personal invitation – issued without consulting cabinet. Public sentiment was beginning to turn against the war, and Ky's visit was met with large demonstrations; opposition leader Arthur Calwell issued a statement calling him a "miserable little butcher". Ky nonetheless handled himself well, and The Bulletin called his visit a "personal triumph".
In March 1966, Holt announced that the 1st Battalion, Royal Australian Regiment, would be withdrawn and replaced by the 1st Australian Task Force, a self-contained brigade-sized unit based at Nui Dat. This effectively tripled the number of Australian troops in Vietnam to around 4,500, and also included 1,500 national servicemen – the first conscripts to serve in the conflict. By the final months of Holt's prime ministership, Australia had over 8,000 personnel stationed in South Vietnam, drawn from all three branches of the Australian Defence Force; the final troop increase was announced in October 1967. Holt "never deviated from his whole-hearted support for American bombing of North Vietnam and the hope that steadily increasing the number of foreign troops deployed to South Vietnam would lead to military victory and a solution to the crisis". John Gorton later said it was "ironical that, being a man of peace, he should have presided over one of the greatest build-ups of military power that Australia has found itself engaged in".
The government's handling of the war initially enjoyed broad public support, and was considered a key contributor to the landslide election victory in 1966 – referred to by some as a "khaki election". By the end of the following year, however, opinion polls were showing that public sentiment had turned against the war, and previously supportive media outlets had begun to criticised Holt's decision-making. He did not live long enough to see the mass demonstrations experienced by his successors. Political opposition to the war was initially led by Opposition Leader Arthur Calwell, who promised a total withdrawal from the conflict and labelled it a "cruel, unwinnable civil war". His replacement, Gough Whitlam, adopted a more pragmatic approach, focusing on policy specifics (particularly the government's apparent lack of an exit strategy) rather than the validity of the war itself.
Holt was the first Prime Minister born in the twentieth century. Holt was an enthusiastic sportsman and avid swimmer, which was a stark differentiation to that of Menzies and the majority of his predecessors and colleagues. Like later successor Bob Hawke, this resonated with positive effect within the electorate. His oratory skills were vastly superior to that of Arthur Calwell whom Holt resoundingly beat in 1966. Holt's rhetorician was, however, considered a match to that of new Labor leader Gough Whitlam. Whitlam himself later said of Holt: