The fate of carbon from long-chain fatty acids and glucose in dairy cows which were fed with protected fat was studied using stable isotope technique. The experiment was carried out on two groups of dairy cows (n=16 in each group) during the first 15 weeks of the lactation period. The cows were fed isoenergetic and isoproteinogenous diets based on corn silage. About 1.8 kg of tapioca starch in the diet of the starch group was substituted by about 0.7 kg of rumen protected fat (Ca salts of palm oil and soybean oil) in the diet of the fat group. The carbon atoms of dietary fat were naturally depleted in 13C as compared to carbon atoms of starch. Daily milk performance and lactose output were significantly (P < 0.05) higher among the cows fed with fat diet. In comparison to the starch group, the enrichment of milk fat with 13C was significantly lower, while that of breath CO2 was significantly higher in the fat group (P < 0.05). This means the fatty acids were incorporated into milk fat in preference to metabolic oxidation. Further studies showed that blood glucose is oxidized to a lower extent and is used for the synthesis of lactose to a higher proportion if the cows were fed with the fat diet. The glucose entry rate into the body glucose pool was not different between the diets. In conclusion, the dietary fatty acids perform a glucose sparing effect and improve the glucose supply for the mammary gland.