Background:The cow urine has been used traditionally for the management of many diseases and also used as bio-enhancer to improve their therapeutic effect. Objective: To prepare and evaluate the anti-obesity potential of distillate cow urine and compare with fresh cow urine against high fat diet induced obesity in Wistar rats. Materials and Methods: Qualitative analysis of fresh cow urine and its distillate was done and used for the evaluation of assessment of anti-obesity parameters like BMI, abdominal circumference, obesity index, atherogenic index, lipid profile analysis and histopathological evaluation after two months daily oral treatment against high fat diet induced Obesity. Results: The treated groups with fresh cow urine and its distillate has reduced BMI, abdominal circumference, obesity index, atherogenic index, total cholesterol, triglycerides, LDL-C and VLDL-C Significantly while increased the levels of HDL-C as compared with control group (P<0.05). The Histopathological evaluation revealed the size of visceral white adipose tissue in treated groups was reduced as compared with control Group. Conclusion: In the present research conclude that fresh cow urine and its distillate both are having significant anti-obesity activity against high fat diet induced obesity in Wistar rats.