BACKGROUND: Mitochondrial DNA copy number (mtDNAcn) is an effective biomarker to evaluate the effects of cationic metals on mtDNA integrity and function. In this study, we explored the relationships between individual and combined urinary metal concentrations and mtDNAcn among workers. METHODS: Blood mtDNAcn and urinary metal concentrations were quantified in a cohort of 328 participants. Restricted cubic spline (RCS) models were applied to explore potential non-linear association, while generalized linear models (GLMs) were utilized to assess the associations between urinary metal levels and blood mtDNAcn. We further explored the combined effects of multiple metals on mtDNAcn through quantile g-computation (GQC) and Bayesian kernel machine regression (BKMR). RESULTS: The dose-response relationship between nickel (Ni) and mtDNAcn exhibited an inverted U-shaped pattern. Notably, GLMs revealed significant reductions in mtDNAcn associated with Ni and silver (Ag) in the second quartile. An overall inverse association between urinary metal mixture and mtDNAcn was observed in the BKMR model when urine dilutions were SG-adjusted. Cre-adjusted lead (Pb) was identified as the primary contributor to decreased mtDNAcn, while SG-adjusted lithium (Li) was indicated as the most substantial contribution to mtDNAcn. CONCLUSIONS: Urinary metal concentrations were associated with decreased mtDNAcn following SG adjustment. Future research should investigate these associations in a larger population with improved calibration techniques.