Context: Blood transfusion is an essential medical procedure conducted with various purposes to provide patients with blood needed. The procedure saves patients’ lives, as blood cannot be manufactured artificially and can only be obtained from human blood sources. Aims: To assess the awareness of, perceptions of, motivations regarding, and barriers to blood donation among a sample of Jazan University students in the Kingdom of Saudi Arabia (KSA). Settings and Design: An observational cross-sectional study was conducted among the undergraduate students of Jazan University. Materials and Methods: Stratified random sampling was used to collect information from 440 students, and a predesigned, structured questionnaire was used to collect data on the study variables. Statistical Analysis: Involved descriptive statistics and inferential statistics. SPSS was used for data analysis. Results: The prevalence of blood donation was 29.0% (95% CI, 25.0–33.3), significantly higher for males at 44.3% (95% CI, 38.3–50.4) than for females at 10.5% (95% CI, 7.3–16.0; P < 0.001), and with odds ratio (OR) = 6.8; [95% CI, 4.1–11.2] than females. Students’ level of knowledge regarding blood donation was low. The main barriers to blood donation were identified as being unqualified for blood donation (57.5%) and risk of contracting infectious disease (48.7%) and the desire to donate in the future to a close friend (38.6%). The main motivations for donating blood were identified as religious reasons (77.5%), altruism (77.5%), and to serve the homeland and meet the call of need (77.5%). Conclusions: The prevalence of blood donation was found to be low, and students’ knowledge regarding blood donation appeared to be lacking. The development of awareness programs among the university’s students will address students’ misconceptions about blood donation and encourage them to join donation campaigns.