This research aimed at analysing the impact of Sport Education on high school students' motivation, considering the effect of gender. The participants were 75 high school students (38 boys and 37 girls, M-age = 16.75, SDage = 0.87) from three physical education classes who completed measures on behavioural regulation, 2x2 achievement goals and social goals in the beginning and the end of the intervention. The intervention programme under Sport Education conditions included twelve 50-minutes lessons, twice per week in regular physical education schedule. The results derived from 2x2 MANOVA showed a significant increase in intrinsic motivation, integrated, identified and introjected regulation, and mastery-approach goals, and responsibility and relationship goals. There was also a significant decrease in amotivation, performance-approach goals and mastery-avoidance goals. With respect to gender, significant differences were found in identified regulation between male and female students at post-test. Moreover, there were significant changes in identified regulation, mastery-approach goals and responsibility goals in male students between pre-test and post-test. For female students, significant differences were found in introjected regulation, amotivation, mastery-avoidance goals, and responsibility and relationship goals between pre-test and post-test. As a whole, Sport Education is a pedagogical model that promotes the high school students' adequate motivational response in terms of self-determination, achievement motivation and social motivation in the sport teaching-learning process in physical education.