BACKGROUND Studies have identified health care providers as an important determinant of vaccination acceptance. However, knowledge and attitudes toward vaccination have not been sufficiently studied in Saudi Arabia, especially among medical students. Therefore, we conducted this study to explore vaccination knowledge and attitudes among medical students at a large Saudi university. METHODS A cross-sectional survey was conducted on 182 Saudi medical students between February 2019 and May 2019. Participants were invited to fill out a self-administered questionnaire assessing knowledge and attitudes toward vaccination. The statistical analysis included descriptive analysis, Chi-square test, independent samples t-test, and analysis of variance (ANOVA). The relationship between knowledge and attitudes was assessed using Pearson's correlation test. RESULTS A total of 182 respondents completed the questionnaires, giving a response rate of 91%. The study included male (52.7%) and female (47.3%) students from study years 2, 3, 4, 5, and 6. The overall mean knowledge score was under average (3.05/9, SD = 1.86) and the respondents showed generally moderate attitudes toward vaccination (Mean = 30.60/45, SD = 6.07). While, there was no sex difference in both the scores on knowledge and attitudes domains, the year of study was significantly associated with the mean knowledge score (F = 6.48, P < 0.01) and attitudes score (F = 7.12, P < 0.01). As predicted, there was a significant linear relationship between vaccination knowledge and attitudes (r = 0.71, P < 0.01). CONCLUSIONS The study revealed generally moderate attitudes of Saudi medical students toward vaccination. However, several knowledge gaps were detected. The implications of the current findings are discussed.