Abstract: Purposes: This study investigated the effects of 1-linoleoyl-3-chloropropanediol (C_18:2-Me) exposure on urinary metabolites in rats using untargeted metabolomics. Methods: Twenty-four male Sprague-Dawley (SD) rats were randomly divided into a normal group (ND), a low-dose group (LD, 7 mg/kg·BW), and a high-dose group (HD, 70 mg/kg·BW). The LD and HD groups received daily intraperitoneal injections for two consecutive days. Untargeted metabolomics analysis based on gas chromatography-mass spectrometry (GC-MS) was performed to analyze rat urine before and after C_18:2-Me exposure. Additionally, histopathological examination of kidney tissue was performed. Results: A total of 33 differential metabolites were screened out between the ND and HD groups, including 25 upregulated and 8 downregulated metabolites. Pathway analysis revealed that C_18:2-Me exposure primarily perturbed β-alanine metabolism, sulfur metabolism, glycine, serine, and threonine metabolism, retinol metabolism, glyoxylate and dicarboxylic acid metabolism, and starch and sucrose metabolism. Histopathological examination showed separation of tubular epithelial cells from the basement membrane and luminal enlargement in the exposed groups. Conclusions: Exposure to C_18:2-Me induced alterations in urinary metabolites and exerted toxic effects on rat kidneys by disrupting several metabolic pathways. Histopathological findings were consistent with the metabolomics results.