One of the most common adverse side effects of cyclophosphamide, a potent anticancer drug which limits its clinical utility is its urotoxicity. The mechanism of cyclophosphamide induced urotoxicity (cystitis) is poorly understood. In the present study the possible role of lysosomal enzymes in cyclophosphamide induced hemorrhagic cystitis was investigated using rat model. Adult female Wistar rats weighing 200-250 gm were used for the study. The rats were administered single intraperitoneal injection of cyclophosphamide at the dose of 150 mg/ kg body wt and sacrificed at various time intervals 6 hr, 16 hr or 24 hr after the dose of cyclophosphamide. The control rats were administered saline alone. The urinary bladder was removed, weighed and used for histological examination and assay of lysosomal enzymes namely acid phosphatase, β glucuronidase and N acetyl glucosaminidase. The weight of the urinary bladder (g/ 100 g body wt) of the drug treated rats was significantly higher compared to that of the untreated rat (0.12± 0.01 Vs 0.064 ± 0.005, P≤ 0.004). Histologically, all the characteristic features of hemorrhagic cystitis and hematuria were observed in the drug treated rats. The extent of damage to the bladder increased with time after treatment. A highly significant increase in protein content (mg/ g wt) was observed in the bladders of the cyclophosphamide treated rats as compared with the control (16.4± 3.0 Vs 6.5 ± 1.7, P ≤0.009). A significant drastic decrease in the activities of the entire lysosomal enzyme studied was observed in the bladders of drug treated rats at 16 hrs and 24 hrs. Acid phosphatase activity was decreased by 437% (P≤0.004); N acetyl glucosaminidase activity was decreased by 403% (P≤ 0.05) and glucuronidase activity by 392% (P≤ 0.009) as compared with the control.Decrease in the activities of lysosomal enzymes in the urinary bladder of cyclophosphamide treated rat may contribute to the urotoxicity of cyclophosphamide.