Chlorpyrifos (CPF) and cadmium (Cd) are widespread environmental pollutants, which are often present in drinking water and foods. However, the combined effects of CPF and Cd were not entirely clear at present. There was also no biomarker available to diagnose the poisoning of the two chemicals at low dose for long-term exposures. In this study, we investigated the change of serum metabolites of rats with subchronic exposure to CPF, Cd, and CPF plus Cd using gas chromatography-mass spectrometerbased metabolomics approach. We performed a stepwise optimization algorithm based on receiver operating characteristic to identify serum metabolite biomarkers for toxic diagnosis of the chemicals at different doses after 90-day exposure. We found that aminomalonic acid was the biomarker for the toxicity of Cd alone administration, and serine and propanoic acid were unique biomarkers for the toxicities of CPF plus Cd administrations. Our results suggest that subchronic exposure to CPF and Cd alone, or in combination at their low doses, could cause disturbance of energy and amino acid metabolism. Overall, we have shown that analysis of serum metabolomics can make exceptional contributions to the understanding of the toxic effects following long-term low-dose exposure of the organophosphorus pesticide and heavy metal.Chlorpyrifos (CPF) is a widely used organophosphorus insecticide with cholinergic toxicity 1,2 . CPF exposure can induce severe toxicity in multiple organs 3 . Cadmium (Cd) is a persistent toxic metal with very long half-life 4 . Chronic Cd exposure induces toxicity in kidneys, liver, and bones 5-8 . Due to their toxicity to different target organs, CPF and Cd pose significant threats to public health.CPF and Cd are widespread environmental toxicants which often coexist in water, air, and soil 3,9 . Given that environmental toxicants are widespread, organisms are seldom exposed to only one chemical. Thus, it is important to study the combined toxicity of multiple toxicants. More and more studies suggest that combined exposure to different chemicals can potentiate the toxicity of single chemical alone 2,10,11 . As the technology advances, new methodologies such as omics approaches are developed, and great progress has been made in the study of mixture toxicology, which increases our ability to predict the potential risks of exposure to mixtures 12 .The acute toxicity of CPF and Cd are well understood 13,14 . We have reported that chronic exposure to CPF and Cd individually and in combination caused neurotoxicity and hepatotoxicity 15,16 . We found that CPF and Cd disturbed energy and amino acids metabolism in the brain and liver. Therefore, in this study, we try to identify the biomarkers in serum, upon exposure to CPF, Cd, and their mixture.Metabolomics is a very useful approach for toxicological studies because it can characterize the changes of metabolite levels induced by toxic chemicals and identify the specific biomarkers for each toxicant 17,18 . In the present study, we aim to use metabolomics analysis in se...