BACKGROUND A body of evidence has suggested bidirectional relationships among gallstone disease (GSD), non-alcoholic fatty liver disease (NAFLD), and kidney stone disease (KSD). However, the results are inconsistent, and studies on this topic in China are relatively few. Our goal is to explore the bidirectional associations among these three diseases through a multicenter study, systematic review, and meta-analysis. AIM To explore the bidirectional associations among these three diseases through a multicenter study, systematic review, and meta-analysis. The results may help to investigate the etiology of these diseases and shed light on the individualized prevention of these three diseases. METHODS Subjects who participated in physical examinations in Beijing, Tianjin, Chongqing in China were recruited. Multivariable logistic regression was employed to explore the bidirectional relationships among GSD, KSD, and NAFLD. Systematic review and meta-analysis were initiated to confirm the epidemiologic evidence from previous observational studies. Furthermore, trial sequential analysis (TSA) was conducted to evaluate whether the evidence was sufficient and conclusive. RESULTS Significant bidirectional associations were detected among the three diseases, independent of potential confounding factors. The pooled results of the systematic review and meta-analysis also corroborated the aforementioned results. The combined evidence from the multicenter study and meta-analysis was significant [pooled odds ratio (OR) = 1.42, 95%CI: 1.16-1.75, KSD -> GSD; pooled OR = 1.48, 95%CI: 1.31-1.67, GSD -> KSD; pooled OR = 1.31, 95%CI: 1.17-1.47, GSD -> NAFLD; pooled OR = 1.37, 95%CI: 1.26-1.50, NAFLD -> GSD; pooled OR = 1.28, 95%CI: 1.08-1.51, NAFLD -> KSD; pooled OR = 1.21, 95%CI: 1.16-1.25, KSD -> NAFLD]. TSA indicated that the evidence was sufficient and conclusive. CONCLUSION The present study presents relatively sufficient evidence for the positive bidirectional associations among GSD, KSD, and NAFLD. The results may provide clues for investigating the etiology of these three diseases and offer a guideline for identifying high-risk patients.