Background: Recent studies suggest that night shiftwork may increase the risk of prostate cancer and elevated serum prostate-specific antigen (PSA) level. The purpose of this study was to verify whether rotating night shiftwork affects serum PSA level. Methods: This study included 3,195 male production workers who work in a large tire manufacturing factory. Serum PSA levels were measured and the data on related factors were obtained. Results: The mean serum PSA level was 0.98 ± 0.79 ng/mL. PSA levels were significantly lower in the younger age group, the obese group, and regular exercise group. PSA levels were lower in night shift workers (n = 2,832) compared to day workers (n = 363), but the difference was not statistically significant. Conclusions: Unlike previous studies, we did not find any evidence that night shiftwork results in an increase in serum PSA levels. Further research and consistent results are needed to elucidate the association between night shiftwork and the effect on the prostate.