Background: Prostate cancer (PCa) is a global burden on public health; it has been ranked as the second common cancer worldwide, with 1.3 million new cases in 2018. Developed countries have a higher incidence than in developing countries. Because of the lack of studies on PCa in Saudi Arabia, our study aims to measure the awareness about PCa and knowledge of PCa screening practices among men in Medina, Jeddah, and Makkah, Saudi Arabia. Methodology: A cross-sectional study conducted among males in Medina, Jeddah, and Makkah, Saudi Arabia, from 2 nd to 10 th July 2020, using a validated questionnaire. The sample size was 1212 participants. Data collected through an online questionnaire. All data analyses performed using (SPSS) software, using the Chi-square test for bivariate analysis. All levels of significance were set at P < 0.05. Results: We collected 1212 completed surveys, with mean ages of 36 years (standard deviation ± 12.77). We found that 77% of participants heard about PCa, and 52.5% heard about PCa screening tests. This study showed that only 10.6% of all participants had good knowledge, 41.9% had fair knowledge, and 47.5% had poor knowledge. Only 3.9% of participants had experienced the prostate-specific antigen test. Conclusion: The study showed that the level of knowledge of PCa is low. Thus, community and individuals should collaborate to increase awareness by having more awareness campaigns, disseminating the information through the media, and encouraging men to do screening tests as indicated. In addition, including more Saudi Arabia cities in future studies is preferable to have more precise outcomes.