Studies indicate that substance use among Ethiopian adolescents is considerably rising; in particular college and university students are the most at risk of substance use. The aim of the study was to assess substance use and associated factors among university students. A cross-sectional survey was carried out among 1040 Haramaya University students using self-administered structured questionnaire. Multistage sampling technique was used to select students. Descriptive statistics, bivariate, and multivariate analysis were done. About two-thirds (62.4%) of the participants used at least one substance. The most commonly used substance was alcohol (50.2%). Being male had strong association with substance use (AOR (95% CI), 3.11 (2.20, 4.40)). The odds of substance use behaviour is higher among third year students (AOR (95% CI), 1.48 (1.01, 2.16)). Being a follower of Muslim (AOR (95% CI), 0.62 (0.44, 0.87)) and Protestant (AOR (95% CI), 0.25 (0.17, 0.36)) religions was shown to be protective of substance use. Married (AOR (95% CI), 1.92 (1.12, 3.30)) and depressed (AOR (95% CI), 3.30 (2.31, 4.72)) students were more likely to use substances than others. The magnitude of substance use was high. This demands special attention, emergency preventive measures, and targeted information, education and communication activity.