It is important to identify priority regions regarding contaminant exposure, especially for environmental epidemiological studies. Whereas very few studies explored spatial variations in concentrations of multiple insecticide metabolites in the general population, as well as their relationship with oxidative stress biomarkers (OSBs). We determined related target analytes in urine samples (n = 850) from 425 healthy adults in six cities of China during both autumn and winter. Eight organophosphorus metabolites (mOPPs), three pyrethroid metabolites (mPYRs), nine neonicotinoid insecticide metabolites (mNNIs), and pentachlorophenol (PCP) were detected in the urine samples. Additionally, linear mixed-effects model and weighted quantile sum model were used to assess the individual and combined effects of the insecticide exposure on selected OSBs. Altogether, 17 out of the 21 analytes were widely detected (73.1-100%). The median specific gravity (SG)-adjusted concentrations for the mOPPs, mPYRs, mNNIs, and PCP were 1.17-4.85, 0.45-0.79, 0.09-1.07, and 0.38 ng/mL, respectively. The mOPPs in Lanzhou (northwest China), mPYRs and mNNIs in Dalian (northeast China), and PCP in Chengdu (southwest China) had the highest concentrations among the six cities. The concentrations of the mNNIs and PCP in urban areas were significantly higher than those in rural areas, while the concentrations of some mOPPs and mPYRs were higher in rural areas. The concentrations of most analytes were higher in autumn than in winter. Hazard quotient of >
1 for chlorpyrifos was observed in 9.2% of the study participants, suggested a potential health risk, while the estimated daily intake values of the other analytes were lower than their chronic reference doses. Many of the insecticide exposure biomarkers were significantly associated with increased OSBs
among them, each interquartile range-fold increase in the insecticide exposure biomarkers was associated with 5.4-19.0%, 4.6-19.4%, and 12.4-83.3% increase in 8-hydroxy-2'-deoxyguanosine, 8-hydroxy guanosine, and 4-hydroxy-2-nonenal-mercapturic acid, respectively. P-Nitrophenol was the main contributor in the association with the increased OSBs. This study found significant variations in insecticide exposure levels among different regions and seasons. The exposure level of chlorpyrifos suggested a potential health risk, and the insecticide mixture exposure was significantly associated with increased OSBs. Further risk assessments are warranted and control measures in the insecticide use are needed to mitigate the potential health risks.