ObjectiveGlyphosate has been ubiquitously present in our living environment due to its efficient herbicidal ability, but its association with the prevalence of kidney stones remains uncertain. This study aims to explore the impact of glyphosate exposure on kidney stones and to investigate the mediating effects of some serologic indicators. Furthermore, we attempt to identify the specific populations at greater risk of exposure. MethodsThis is a cross-sectional study of the U.S. adult population examining the association between glyphosate exposure and kidney stones based on data from the 2013–2018 National Health and Nutrition Examination Survey (NHANES). We implemented multi-model-adjusted logistic regression and smoothed curve fitting to explore the connection between them. Further subgroup analyses were conducted to confirm the magnitude of exposure risk in specific populations. Mediation effects analysis served to provide insight into the underlying mechanisms of the link. ResultsA total of 4302 participants' health data were ultimately analyzed, and the prevalence of kidney stones was 10.85 %. Participants with the highest urinary glyphosate(uGLY) content(Q3) had a higher prevalence of kidney stones compared with participants with the lowest uGLY content(Q1) (OR=1.70, 95 %CI: 1.10–2.63). Smoothed curve fitting revealed a linear positive association between ln-transformed uGLY and kidney stones (OR=1.21,95 %CI:1.08–1.37, LLR=0.291), and this exposure-outcome effect was at greater risk in men (OR=1.24,95 %CI: 1.05–1.46), non-Hispanic whites (OR=1.29, 95 %CI: 1.09–1.53), and hypertensive groups (OR=1.23,95 %CI: 1.05–1.44). Serum biochemical markers HDL, ALP, and serum glucose partially mediated the correlation between glyphosate and kidney stones (2.44–4.20 %). ConclusionGlyphosate exposure is significantly associated with the prevalence of kidney stones. In men, non-Hispanic whites, and hypertensive populations, the management of glyphosate exposure should be emphasized, and appropriate protective strategies may be beneficial in reducing the burden of kidney stones. More high-quality clinical inquiries and animal toxicology experiments are still required to verify the reliability of our findings and their underlying mechanisms.