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Few studies have examined the relationship between lipid metabolism and kidney stone formation, particularly the role of key lipid regulatory factors in kidney stone formation. We evaluated the effect of the lipid regulatory factor‐peroxisome proliferator‐activated receptor alpha on the formation of renal stones in mice by injecting them with glyoxylate followed by treatment with either a peroxisome proliferator‐activated receptor alpha agonist fenofibrate or an antagonist GW6471 (GW). Liquid chromatography coupled with trapped ion mobility spectrometry‐quadrupole‐time‐of‐flight mass spectrometry‐based lipidomics was used to determine the lipid profile in the mouse kidneys. Histological and biochemical analyses showed that the mice injected with glyoxylate exhibited crystal precipitation and renal dysfunction. Crystallization decreased significantly in the fenofibrate group, whereas it increased significantly in the GW group. A total of 184 lipids, including fatty acyls, glycerolipids, glycerophospholipids, and sphingolipids differed significantly between the mice in the model and control groups. Peroxisome proliferator‐activated receptor alpha activity negatively correlated with glyoxylate‐induced kidney stone formation in mice, which may be related to improved fatty acid oxidation, maintenance of ceramide/complex sphingolipids cycle balance, and alleviation of disorder in phospholipid metabolism. [ABSTRACT FROM AUTHOR]