2001: three-year-old career
Galileo was conditioned for a middle-distance campaign and aimed at Derby trial races in late spring. On his debut, he was 1/3 favourite for the Listed Ballysax Stakes over a mile and a quarter at Leopardstown, which he won "easily" by three and a half lengths. Runner-up Milan went on to win the St Leger and finish second in the Breeders' Cup Turf, while the third-placed Vinnie Roe won four consecutive Irish St. Legers. O'Brien was satisfied with the performance, especially as he believed the colt was some way from peak fitness: ("[my horses]... are as big as bulls") and likely to improve.
Galileo was stepped up in class for the Group Three Derrinstown Stud Derby Trial at Leopardstown in May. The odds-on favourite, he was ridden for the first and only time by Seamie Heffernan, as Kinane was riding Milan in the Group One Prix Lupin on the same day. Galileo beat the John Oxx-trained Exaltation by one and a half lengths. Although Heffernan put Galileo under pressure before going clear, the colt's Derby odds were cut from 8–1 to 5–1.
At Epsom, Galileo started 11/4 joint-favourite with 2,000 Guineas winner Golan. The 2001 Epsom Derby field was strong, with Tobougg, the previous year's champion two-year-old, and the winners of most of the major Derby trials, including Mr Combustible (Chester Vase), Dilshaan (Dante Stakes) and Perfect Sunday (Lingfield Derby Trial), all taking part. Two furlongs from the finish, Galileo moved past Mr Combustible and quickly went clear to record an "impressive" three and a half length victory over Golan, Tobougg and Mr Combustible. Following the race, Timeform assigned a figure of 130 to Galileo, making him, in their opinion, the best Derby winner for ten years. Kinane was reported to have called Galileo the best horse he had ridden: "There is no weakness to him... He is a dream to ride." It was the first Derby winner for O'Brien and the first Derby winner Sadler's Wells had sired.
In the Irish Derby at The Curragh, three weeks later, Galileo was 4/11 favourite, with Golan and Exaltation the only other horses starting at less than 20/1. Although eased down in the closing stages, the colt won by four lengths from the Derby Italiano winner Morshdi, with Golan another four lengths back in third. Kinane, who was winning his first Irish Derby in seventeen attempts, said that he was always in "complete control", while Morshdi's jockey, Philip Robinson, described the winner as "a freak".
Galileo was then tested against older horses in the King George VI and Queen Elizabeth Diamond Stakes at Ascot in July. The betting suggested a match between Galileo (1/2 favourite) and the Godolphin five-year-old Fantastic Light (7–2), who had won Group One races in four different countries. Such were the reputations of these two horses that the third choice in the betting, the French Derby winner Anabaa Blue, started at 18/1. Kinane took Galileo to the lead two furlongs out, but Frankie Dettori challenged hard on Fantastic Light as Galileo won by two lengths.
Galileo returned to Leopardstown in September for a much-anticipated Irish Champion Stakes against Fantastic Light again, this time over the shorter distance of one and a quarter miles. O'Brien's Ice Dancer was to act as pacemaker and ensure a stamina test. The plan failed, as the rest of the riders ignored Ice Dancer, who went ten lengths clear before stopping abruptly in the straight. Fantastic Light and Galileo raced side by side, with the former winning by a head. The race has been described as one of the most exciting and memorable in the modern history of the sport. O'Brien praised the winner but felt that he himself had "messed up" tactically.
For his final race, Galileo was sent to Belmont Park for the Breeders' Cup Classic. He would be racing on dirt for the first time, although he was prepared by exercising on the artificial surface at Southwell. In the Classic, Galileo finished sixth of the fourteen runners behind Tiznow.
"With the benefit of hindsight", O'Brien later recalled, "it was an unrealistic target to ask him to do that after having such a tough season and racing against the older horses, but it was the belief that was in him, the belief that everyone had in him, that we thought it could be possible that it could happen. I remember when he came in, he was after trying so hard he was almost crying. He was so genuine."
Galileo's retirement was announced immediately after the race.