INTRODUCTION: University students in Rwanda are at high risk for HIV, yet they have a low uptake of HIV self-testing, which is crucial for HIV diagnosis and prevention. This study investigated their knowledge, behaviors, and perceptions towards HIV self-testing, highlighting the barriers and opportunities whose consideration is necessary for the improvement of HIV self-testing uptake in this population. METHOD: A concurrent mixed-method design was used, and it involved 424 students from five universities across Rwanda. Quantitative data was collected through surveys, and descriptive statistics were performed. Chi-square tests were performed, and sociodemographic variables were stratified against the awareness of HIV self-testing and HIV self-testing for the past 12 months variables. Qualitative data was collected through in-depth interviews and focus group discussions using interview guides developed based on the Health Belief Model (HBM) framework; data was then analyzed thematically. RESULTS: The mean age was 23 (IQR: 21; 24), with 51.2% (n = 214/424) females. 64.7% (n = 261/424) of students had never heard of HIV self-testing, yet 37.74% (160/424) were sexually active. Among sexually active students, 17.87% were aware of HIV self-testing, but 35.82% had never used it. The reported perceived HIV self-testing barriers include high cost, unavailability of testing kits, lack of awareness, misinformation, and absence of post-test counseling. However, some HIV self-testing opportunities, like the availability of testing kits and motivating factors for university students to test, were also reported. CONCLUSION: Although university students reported the needs and benefits of HIV self-testing, uptake remains low due to misinformation, unawareness, unavailability, and the high cost of HIV self-testing kits. Increasing awareness, availing HIVST kits, and addressing the other reported barriers to HIV self-testing, is essential for the achievement of the universal goal of HIV status awareness among university students.