Background and Aim: The influence of metabolic dysfunction‐associated fatty liver disease on gallstone development remains unclear. We aimed to investigate the longitudinal association between metabolic dysfunction‐associated fatty liver disease and gallstone development in both men and women. Methods: This observational cohort study included 5398 patients without gallstones who underwent > 2 health check‐ups between April 1, 2014, and March 31, 2020. A generalized estimation equation model was used to analyze the association between metabolic dysfunction‐associated fatty liver disease and gallstone development according to repeated measures at baseline and most recent stage. Results: After adjustment, the odds ratios of metabolic dysfunction‐associated fatty liver disease for gallstone development in men and women were 3.019 (95% confidence interval [CI]: 1.901–4.794) and 2.201 (95% CI: 1.321–3.667), respectively. Among patients aged ≥ 50 years, the odds ratio for gallstone development was significantly enhanced with increasing metabolic dysfunction‐associated fatty liver disease component numbers in both sexes; however, no significance was observed in those aged < 50 years. Other significant risk factors for gallstone development were age (odds ratio: 1.093, 95% CI: 1.060–1.126) and waist circumference (odds ratio: 1.048, 95% CI: 1.018–1.079) in men and age (odds ratio: 1.035, 95% CI: 1.003–1.067) and current smoking (odd ratio: 5.465, 95% CI: 1.881–15.88) in women. Conclusion: Although the risk factors for gallstone development differed between sexes, metabolic dysfunction‐associated fatty liver disease was common. Paying attention to an increase in the number of metabolic dysfunction‐associated fatty liver disease components in patients aged ≥ 50 years is important for gallstone prevention. [ABSTRACT FROM AUTHOR]