Grounded in self-determination theory, this study aimed first to examine differences in motivation for physical activity (PA) and habitual PA levels between adolescents and university students in Spain. The second aim was to examine differences in the degree of association between their intrinsic motivation and amotivation for PA and habitual PA levels in both samples. We studied 2,699 students (1,833 high school and 866 university; mean age = 18.83, standard deviation = 4.12 years) who completed the International Physical Activity Questionnaire-Short Form and two dimensions of the Behavioral Regulation in Exercise Questionnaire. High school students, relative to university students, self-reported significantly higher intrinsic motivation and higher PA levels for each type of PA intensity (i.e., walk, moderate, and vigorous); high school students also self-reported significantly lower amotivation than university students. The degree of association between the total metabolic equivalents of task and intrinsic motivation was higher among high school (versus) university students. This study shows the need to design intervention strategies to enhance PA levels and intrinsic motivation in university students.