On January 7, 2006, Mustain was the starting quarterback for the West team in the U.S. Army All-American Bowl in San Antonio, Texas, where he was awarded the Hall Trophy as National High School Player of the Year.
Mustain was the top quarterback in the nation coming out of high school in 2006, and was considered a better prospect than eventual Heisman-winner Tim Tebow. In recognition of his accomplishments, Springdale Mayor Jerre Van Hoose declared December 6, 2005 "Mitch Mustain Day" in the city.
Mustain enrolled at the University of Arkansas on July 10, 2006 and, in his first game on September 2, 2006, replaced starting quarterback Robert Johnson in the fourth quarter of an eventual 50–14 loss to the USC Trojans. He immediately put together an 80-yard touchdown drive against the Trojan defense. The following day, Arkansas head coach Houston Nutt named Mustain as the starting quarterback for the next game against Utah State, moving Johnson to wide receiver.
Mustain accumulated a record of 8–0 as a true freshman starter, including a 27–10 win on the road over the #2 ranked Auburn Tigers on October 7, 2006, helping guide the Razorbacks to 11th in the college football rankings. The offense was oriented around what was considered among the nation's best running back tandems, Darren McFadden (who would be the runner-up for the Heisman Trophy), Peyton Hillis, and Felix Jones; as a result, Mustain's statistics did not match his high school numbers in a more pass-oriented offense.
A turning point occurred on November 4, 2006 in No. 11 Arkansas' 26–20 win over South Carolina, when Mustain was relieved after one series by sophomore Casey Dick, who led the Razorbacks for 228 yards and a touchdown. The day after the game, Dick was named the starting quarterback for an upcoming game against #13 Tennessee, with Coach Nutt citing "experience" as the primary reason for the change.
Mustain did not play in the next three games and entered for one series in the final game of the regular season, the SEC Championship game, on December 2, 2006, against the Florida Gators.
On December 20, 2006, Nutt announced that Dick would start for the Razorbacks in the January 1, 2007 Capital One Bowl against the Wisconsin Badgers; however, he stated that Mustain would also enter the game in the third series. The game resulted in a 17–14 defeat for Arkansas.