Background: Physical activity has been hypothesized to reduce the risk of gallbladder disease (gallstones, cholecystitis, cholecystectomy); however, results from epidemiological studies have not always shown statistically significant associations. We conducted a systematic review and meta-analysis to clarify the presence and strength of an association between physical activity and gallbladder disease risk. Methods: PubMed and Embase databases were searched for studies of physical activity and gallbladder disease up to 9th of January 2015. Prospective studies reporting relative risk (RR) estimates and 95% confidence intervals (CIs) of gallbladder disease associated with physical activity were included. Summary RRs were estimated using a random effects model. Results: Eight studies including 6958 cases and 218,204 participants were included. The summary RR for the highest versus the lowest level of physical activity was 0.75 (95% CI: 0.69–0.81, n = 8) and there was no evidence of heterogeneity, I2 = 0%). In the dose-response analysis the summary relative risk per 20 MET-hours of activity was 0.85 (95% CI: 0.80–0.90, I2 = 0%, n = 2) for leisure-time physical activity, 0.83 (95% CI: 0.76–0.90, I2 = 0%, n = 2) for vigorous physical activity, and 0.86 (95% CI: 0.76–0.98, I2 = 0%, n = 2) for nonvigorous physical activity. Conclusion: Our analysis confirms a protective effect of physical activity on risk of gallbladder disease.