The International Agency of Research in Cancer (IARC) has recently confirmed shift work as a type 2A carcinogen. The results presented in published epidemiological studies regarding prostate cancer are inconsistent and the association remains controversial. The aims of this study were: (a) to investigate the possible association between shift work and prostate cancer incidence, identifying possible sources of heterogeneity; and (b) to analyze the potential effect of publication bias. A search for cohort and case-control studies published from January 1980 to November 2019 was conducted. The quality of the articles was assessed using the Newcastle–Ottawa Scale. Pooled OR were calculated using random-effects models. Heterogeneity was evaluated using Cochran’s Q test and data were stratified by potential sources of heterogeneity. Publication bias was analyzed. Eighteen studies were included. No association was found between rotating/night-shift work and prostate cancer, pooled OR 1.07 (95%CI 0.99 to 1.15), I2 = 45.7%, p = 0.016. Heterogeneity was eliminated when only cohort studies (pooled OR 1.03; 95%CI 0.96 to 1.10; I2 = 18.9%, p = 0.264) or high-quality studies (pooled OR 0.99; 95%CI 0.89 to 1.08; I2 = 0.0%, p = 0.571) were considered. A publication bias was detected. An association between shift work and prostate cancer cannot be confirmed with the available current data. Future analytical studies assessing more objective homogeneous exposure variables still seem necessary.