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BACKGROUND: Some plant extracts can prevent kidney stone formation in a dose-dependent manner. In our study, we aimed to investigate the protective role of Cannabis sativa aqueous extraction on calcium oxalate formation in ethylene glycol-induced rats. MATERIALS AND METHODS: To evaluate anti-urolithiasis, variations of the main risk factors (citrate, phosphorous, calcium, and Ph) have been evaluated in 24-hour urine samples of rats one day before the end of the experiment. Rats were divided into 4 groups: group 1 was administered regular drinking water; group 2 was administered 1% ethylene glycol in drinking water, group 3 was administered 100 mg/kg of Cannabis sativa extract for oral administration by gavage along with drinking water containing 1% ethylene glycol; and group 4 was administered 200 mg/kg of Cannabis sativa extract by gavage along with drinking water containing 1% ethylene glycol. Finally, histopathological slides from the kidney were also evaluated. RESULTS: Results showed that 100 mg/kg and 200 mg/kg doses of Cannabis sativa extract significantly reduced the mentioned risk factors in comparison with ethylene glycol-treated rats (p<0.05).  CONCLUSION: Histopathological slides showed reduced calcifications with extract treatment at 200 mg/kg of Cannabis sativa. Thus, this antioxidant herb can prevent kidney stone formation. These findings pave the way for new therapy.