Purpose To investigate the anti-urolithiatic effect of cow urine ark (medicinal distilled cow urine) on ethylene glycol (EG) induced renal calculi. Materials and Methods 36 male Wistar rats were randomly divided into 6 equal groups. Group I animals served as vehicle control and received distilled water for 28 days. Group II to VI animals received 1% v/v EG in distilled water for 28 days. Group II served as EG control. Group III and IV (preventive groups) received cow urine ark orally for 28 days in doses of 1 mL/kg and 2 mL/kg, respectively. Group V and VI (treatment groups) received 1 mL/kg and 2 mL/kg cow urine ark orally, respectively from 15th to 28th days. 24-hour urine samples were collected on day 0 and 28. Urine volume and oxalate levels were measured. On day 28, blood was collected for biochemical parameters. Animals were sacrificed and kidneys were harvested, weighed and histopathologically evaluated for calcium oxalate (CaOx) crystals. To calculate the percentage of inhibition of mineralization, simultaneous flow static in-vitro model was used. Results EG significantly increased urine oxalate, serum creatinine, blood urea level; kidney weight and CaOx deposits. Provision of cow urine ark resulted in significantly lower levels of urine oxalate, serum creatinine, blood urea and CaOx depositions as compared to Group II. (p value < 0.05) It also significantly restored kidney weight. (p value < 0.05) Cow urine ark inhibited 40% and 35% crystallization of CaOx and calcium phosphate, respectively. Conclusion Cow urine ark is effective in prevention and treatment of EG induced urolithiasis in Wistar rats.