Background: Substance use is a major mental health concern among university students. It may result in behavioral and academic problems, psychiatric disorders, and infectious diseases. Thus, this study investigated the risk and protective factors of substance use among Iranian university students. Methods: This was a cross-sectional study. A number of 7330 students were selected from 30 universities in Iran. The participants completed a researcher-designed questionnaire. It measured life time, previous year and previous month’s substance use, demographic characteristics, and a body of risk and protective factors including, religious beliefs, self-esteem, stress and psychological pressure, sensation seeking, attention seeking, anger and aggression, depression and anxiety, parents’ positive attitude towards substance use, lack of intimacy between family members, plus substance use, smoking cigarettes or hookah, alcohol consumption, and prescribed medications use by their family members, easy access to illegal drugs, peers’ positive attitude towards substance use, peers’ drug use, perceived prevalence of substance use among students, and negative attitude toward university. The data were analyzed using descriptive statistics and multivariate logistic regression analysis. Results: Participants’ anger and aggression, depression and anxiety, participants’ positive attitude towards substances, low level of religious beliefs, peers and family member’s substance use, and parent’s positive attitude towards substance significantly and strongly predicted using cigarette/hookah, alcohol, hard drugs, and prescribed medications. Having a negative attitude toward university significantly predicted using all types of substance (except for prescribed medications). Low self-esteem predicted using cigarette/hookah, and alcohol use. Perceived availability of illegal drugs predicted hard drugs and prescribed medications’ consumption. Finally, peers’ positive attitude toward drugs anticipated cigarette/hookah use. Conclusion: Prevention programs are most needed among Iranian students. They should be comprehensive in nature and focus on students’ psychoeducation about substances and their related negative consequences, plus promotion of students’ life skills, and integrate family- and peer-based preventive interventions.

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