The present study was designed to evaluate the antiurolithiatic effect of PHYMIN-22 against ethylene glycol-induced urolithiasis in rats. Healthy Albino male rats with 200-230g body weight were randomly divided into five groups, each with 5 animals, control group, EG group (0.75%), PHYMIN-22 treatment group (0.75% EG 14days and 100mg/kg PHYMIN-22 next 14days), PHYMIN-22 drug control group (100mg/kg) and cystone treatment group (0.75% EG 14days and 750mg/kg cystone next 14days). Biochemical testing was adopted for measuring the blood and urine parameters, as well as the level of antioxidants including superoxide dismutase (SOD), Catalase (Cat), Glutathione peroxidase (GPx) and glutathione (GSH) in kidney tissues. Hematoxylin and eosin (HE) staining was utilized to observe the histopathological changes in the kidney tissue. End of the experiment the PHYMIN-22 treatment reduced the urine and serum calcium (p < 0.01; p < 0.01), oxalate (p < 0.01; p < 0.01), phosphate (p < 0.01; p < 0.01), uric acid (p < 0.001; p < 0.001), protein (p < 0.001; p < 0.001), and creatinine (p < 0.001; p < 0.001) respectively, serum indicators ALT (p < 0.001) and AST (p < 0.001) level and non-enzymic antioxidant GSH (p < 0.001) compared to EG induced urolithiasis animals (Diseased control group). PHYMIN-22 treatment significantly increased urine volume, pH, and body weight, and antioxidants include CAT (p < 0.001; p < 0.001), SOD (p ˃ 0.05; p < 0.05), and GPX (p < 0.01; p < 0.001) compared to Diseased control group animals. The effect of PHYMIN-22 on EG-induced urolithiasis animals could be by improving kidney function, normalizing the urine and serum parameters, maintaining the kidney antioxidants, eliminating crystal deposition, and excretion of unwanted ions from the kidney and urinary tract.