Canadian 24 h movement guidelines recommend engaging in >150 min/week of moderate–vigorous-intensity physical activity and ≤8 h/day of sedentary time. Half of Canadian post-secondary students do not meet physical activity or sedentary time guidelines. This pan-Canadian study aimed to (1) identify commonly cited motivators/barriers to exercise, and (2) determine which motivators/barriers were most influential for attaining physical and sedentary activity guidelines. A total of 341 respondents (279 females, 23 ± 4 years old, 53% met activity guidelines, 49% met sedentary guidelines) completed an online survey regarding undergraduate student lifestyle behaviours. Improved physical health (74% of respondents), mental health (67%), physical appearance (60%), and athletic performance (28%) were the most common motivators to exercise. The most common barriers were school obligations (68%), time commitments (58%), job obligations (32%), and lack of available fitness classes (26%). Students citing improved athletic performance (odds ratio (OR) = 1.94, p = 0.02) were more likely to adhere to activity guidelines, while those who selected physical health (OR = 0.56, p = 0.03) and physical appearance (OR = 0.46, p = 0.001) as motivators were less likely to meet activity guidelines. Students who cited school obligations as a barrier were less likely (OR = 0.59, p = 0.03) to meet sedentary guidelines. The motivators and barriers identified provide a foundation for university-led initiatives aimed at promoting physical activity and reducing sedentary time among undergraduate students. Strategies that positively re-frame students’ physical health and appearance-based motivations for exercise may be particularly useful in helping more students achieve national activity recommendations.