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Objective Atrazine, an herbicide widely used in US agriculture, has been associated with kidney cancer and non-malignant kidney disease. However, the potential mechanisms underlying these associations remain unclear. We evaluated atrazine use and biomarkers of kidney function and nephrotoxicity among male farmers in the Biomarkers of Exposure and Effect in Agriculture Study.Methods Our investigation included three groups of farmers defined based on atrazine use: (1) both recent (last 3 months) and past use (continuous users; n=83); (2) high lifetime use (≥178.5 lifetime days) but no recent use (former high users; n=88) and (3) never/low lifetime use (<50 lifetime days) and no recent use (never/low users; n=75). Biomarkers were measured in serum (creatinine, cystatin C, urea nitrogen, uric acid) and urine (kidney injury molecule-1 (KIM-1), albumin, creatinine). The 2021 CKD-EPI creatinine-cystatin C equation was used to calculate estimated glomerular filtration rate (eGFRcr-cys). We estimated the percentage difference (95% CI) in each marker across groups using multivariable linear regression.Results Compared with farmers with never/low atrazine use, continuous users had statistically significantly lower eGFRcr-cys (−9.4%; 95% CI –16.1% to –2.2%) and higher serum creatinine (9.4%; 95% CI: 1.6% to 17.9%) and cystatin C (10.8%; 95% CI 2.0% to 20.4%); no associations with these markers were observed for the former high users. We also observed higher uric acid for both former high and continuous users and lower urea nitrogen for former high users, although these associations were not statistically significant. Urine albumin-creatinine ratio and KIM-1 levels did not differ across groups.Conclusions Our findings add to the evidence that continued atrazine use is associated with diminished kidney function.