Abstract: The aim of the present study was to determine the metabolic changes and possible toxic mechanisms of ketamine-associated bladder toxicity. Twenty-four male Sprague-Dawley (SD) rats were randomly allocated into a control group, a low-dose group and a high-dose group. The behavior of these rats was observed every day. In addition, the weight, 2 h urinary frequency and organ coefficient of the bladder were measured. Serum IL-6 and TNF-α levels were measured using an enzyme-linked immunosorbent assay (ELISA). Urinary metabolites were analyzed using gas chromatography-mass spectrometry (GC-MS). This research was approved by the Ethics Committee of the Animal Experiment Center of Southwest Medical University (No. 201901-98). After 12 weeks of administration, the frequency of 2 h urination and the bladder mass index were significantly different in the low-dose and high-dose groups compared with the control group. Serum IL-6 and TNF-α levels were higher than those of the control group (P<0.05). Bladder HE staining showed that long-term administration of ketamine could induce cystitis. The concentrations of the three common differential metabolites, including 3-aminoisobutyric acid, citric acid and uric acid in the low-dose and the high-dose groups were increased compared with those in the control group. This study indicates that 3-aminoisobutyric acid, citric acid and uric acid and their related metabolic pathways may be closely related to ketamine-associated bladder toxicity.