Using her father (a bus driver)’s redundancy money, and working with her brother, Brian Souter, and her first husband, Robin Gloag, she established the Stagecoach Group in 1980, running buses from Dundee to London. Expansion continued and in the early 1990s, Stagecoach acquired National Bus Company operations in Cumberland, Hampshire, East Midlands, Ribble, Southdown and the United Counties. Stagecoach bought further bus operations in Scotland, Newcastle and London, with Manchester being added in 1993.

Gloag has owned Beaufort Castle near Inverness since 1995, and Kinfauns Castle, near Perth since 2004. She has attempted to block off private access at Kinfauns in a high-profile case. On 12 June 2007 she was successful in gaining a court ruling that she was legally entitled to bar the public from a swath of woodland in the grounds of Kinfauns Castle.

Her ex-husband, Robin Gloag, father of her late son, Jonathan, was killed in a car crash on 6 December 2007. Their son, Jonathan, hanged himself in 1999, aged 28. In 1990, Ann Gloag married David McCleary, a widower and a successful owner of multiple businesses, who was previously an organist and minister at the local Church of the Nazarene.

In November 2013, Gloag took ownership of Kent International Airport, also known as Manston Airport, for the sum of £1. Gloag’s co-director is Pauline Bradley, a corporate lawyer and former head of joint ventures at Bank of Scotland, described by The Herald as “one of Scotland’s most powerful women”.

Despite assurances to staff on the long-term investment in the airport, management announced a consultation on closure in April 2014. Uncertainty about the airport’s future led flight operators that were using Manston to leave, notably KLM, which was running a twice-daily service to Amsterdam Schiphol. A number of bids were forthcoming during the consultation period to buy and run the airport, but, on 15 May 2014, Manston was closed with the loss of 144 jobs in the airport and an unknown number in the surrounding area. Gloag did not appear publicly or give a reason for the airport’s closure or her refusal to sell.

As of 2016, Gale and pressure groups including Save Manston Airport and Why Not Manston? continue to campaign for the reopening of the airport and have opposed alternative uses. In May 2014, Prime Minister David Cameron, in answer to a question from Gale in the Houses of Commons, stated that the future of Manston was the responsibility of the airport owner, but the Government was in negotiation with Gloag. In July 2017 planning inspectors rejected an appeal stating that the site was protected for aviation use under EC4 regulations.

Ann Gloag was made a Dame Commander of the Order of the British Empire in the 2019 New Year Honours list. Ann, who was previously awarded an OBE in 2004, has received her damehood in recognition of her extensive business and charitable work in the UK and overseas. The Gloag Foundation was set up by Ann Gloag to support projects that prevent or relieve poverty and encourage the advancement of education, health and religion in the UK and overseas.