Abstract: Objective: To investigate the interaction and joint effect of serum melatonin (MT) and vitamin D (Vit D) on the risk of overweight and obesity, and to explore the mediating role of metabolic indicators. Methods: This cross-sectional study enrolled 3, 739 health examinees, who were categorized by body mass index (BMI) into non-overweight/obesity (n=2,029) and overweight/obesity (n=1,710) groups, and subsequently stratified by median serum MT levels and clinical Vit D status (sufficient, insufficient/deficient). Multiple linear regression and logistic regression models were employed to analyze the independent and joint effect of MT and Vit D on BMI and the risk of overweight/obesity. Subgroup analyses were conducted to examine potential effect heterogeneity across different populations, and mediation analysis was performed to assess the mediating effect of metabolic parameters. Results: Multivariate-adjusted models revealed a significant multiplicative interaction between serum MT and Vit D on BMI (interaction term, β=-0.47, P=0.001). Analysis of their joint effect demonstrated that compared to the“low MT + insufficiency/deficiency Vit D”group, the“high MT + Sufficient Vit D”group was significantly associated with a lower BMI (β=-0.64, 95% CI: -0.97 to -0.31, P＜0.001) and a reduced risk of overweight/obesity (OR=0.76, 95% CI: 0.61 to 0.93, P=0.010). Furthermore, a significant dose-response relationship was observed for this joint effect. Subgroup analyses indicated that this joint protective association was particularly pronounced among older adults, individuals with type 2 diabetes mellitus, and those with hypertension. Mediation analysis further identified total cholesterol, triglycerides, low-density lipoprotein cholesterol, fasting blood glucose, and the triglyceride-glucose (TyG) index as significant mediators of the joint effect on BMI, accounting for 10.2%, 33.4%, 5.6%, 18.3%, and 57.0% of the total effect, respectively. Conclusion: Serum MT and Vit D exhibit a joint protective effect against the risk of overweight and obesity. Maintaining relatively higher MT and sufficient Vit D levels may represent a potential strategy for preventing overweight and obesity. This protective joint effect is likely mediated through the improvement of insulin resistance and lipid metabolism.