The aim of this study was to determine frequency and motives for participation in physical activity (PA) and to explore the relationships between PA and self-reported levels of depression, anxiety and stress. The study sample included 290 students (100 males and 190 females) from various universities in Republic of Croatia. Research took place in period between December 2014 and February 2015. In the online survey, the participants fulfilled the General questionnaire which was created for this research, and Croatian versions of EMI-2 (The Excercise Motivation Inventory 2) and DASS-21 questionnaire (Depression, Anxiety, Stress Scale). The results show a statistically significant difference in frequency of PA participation between males and females, in favor of males. Also, there was statistically significant difference between males and females in motives for PA participation. Males obtained significantly higher results in following motives: Affiliation, Competition, Enjoyment, Social recognition and Strength. On the other hand, females reported significantly higher results in motives of Appearance, Nimbleness, Maintaining/improving health and Weight management motive. There was no other significant difference between males and females regarding their motives for participation in PA. In comparison to their physically insufficiently active or inactive colleagues, male students who are physically active minimally 3 times per week obtained statistically significant lower levels of depression. No statistically significant difference was found in measures of stress and anxiety in male sample. Contrary to our expectations, female students who reported low and those who reported high levels of PA didn„t differ according to the levels of depression, stress, and anxiety. The results of this study could be used in a process of creating sports programs, depending on the gender of students.