Background: In early post-partum buffaloes, feed intake is generally insufficient to meet the energy demand for milk production; as a consequence, body reserves are mobilized which can result in metabolic disorders and poor production performance. In order to enhance energy intake and reduce the detrimental effects of a negative energy balance, buffalo diets may be supplemented with lipids/vegetable oil. Some reported benefits of vegetable oil supplementation include increased energy concentration in the diet, reduced supply of rapidly fermentable carbohydrates, and better productive performance. Sunflower (Helianthus annuus L.) is an important oilseed crop in India. Sunflower seed contains about 48-53 percent of edible oil. Recent studies demonstrated that sunflower oil supplementation improved the milk production and nutritional quality of milk fat which is beneficial to human health. Therefore, the present study was undertaken to investigate the effect of sunflower (Helianthus annuus L.) oil supplementation on production performance of lactating Mehsana buffalo. Methods: Three experimental groups were as, CON: fed on basal diet (Control), SO125: fed basal diet supplemented with 125 ml of sunflower oil/animal/day and SO250: fed basal diet supplemented with 250 ml of sunflower oil/animal/day for a period of 90 days. Result: There was no difference (P greater than 0.05) in DM intake of lactating buffaloes among the dietary treatments. Dietary inclusion of sunflower oils improved milk yield by 17.9 to 20.7%, 6% FCM yield by 16.4 to 22.9% and ECM yield by 16.0 to 23.0% in SO125 and SO250 groups, respectively as compared to the CON. There were no differences (P greater than 0.05) in milk composition and yields of milk components. The milk fatty acid percentages of C20:0 and C24:0 were significantly decreased due feeding of sunflower oil as compared to the CON. Feeding of sunflower oil did not influence milk fat short chain, medium chain, saturated, unsaturated and polyunsaturated fatty acids in lactating Mehsana buffaloes. The serum concentrations of glucose, total proteins, albumin, urea, creatinine, triglycerides, ALT and AST were not affected (P greater than 0.05) by supplementation of sunflower oil, however, serum cholesterol concentration was significantly (P less than 0.01) increased in SO125 and SO250 groups as compared to the CON group.