Display omitted •Urines from female and male residents under cadmium exposure were investigated by mass spectrometry-based metabolomics.•Biomarkers and metabolic pathways related to cadmium-induced nephrotoxicity were characterized.•This study might provide information to reflect the body burden and help improve health policy for risk assessment. Cadmium is a well-known hazardous pollutant that mainly comes from dietary, tobacco and occupational exposure, posing threat to kidney. However, there is still a lack of systematic study on metabolic pathways and urinary biomarkers related to its nephrotoxicity under cadmium exposure for both females and males. In this study, a mass spectrometry-based metabolomics investigation of a cohort of 144 volunteers was conducted to explore sex-specific metabolic alteration and to screen biomarkers related to cadmium-induced nephrotoxicity. When the concentration of urinary cadmium increased, creatine pathway, amino acid metabolism especially the tryptophan metabolism, aminoacyl-tRNA biosynthesis, and purine metabolism were primarily influenced regardless of the gender. Also, the most specific biomarkers linked with nephrotoxicity based on the statistical analysis were detected including creatine, creatinine, l-tryptophan, adenine and uric acid. The study outcome might provide information to reflect the body burden and help improve health policy for risk assessment.