Objective: To evaluate soy sauce oil (a by-product of making whole soybean soy sauce) as a new dietary lipid source, a large amount of soy sauce oil was administered into the rumen of dairy cows. Methods: Four Holstein dairy cows fitted with rumen cannulae were used in a 56-day experiment. Ruminal administration of soy sauce oil (1 kg/d) was carried out for 42 days from day 8 to day 49 to monitor nutritional, physiological and production responses. Results: Dry matter intake and milk yield were not affected by soy sauce oil administration, whereas 4% fat-corrected milk yield and the percentage of milk fat decreased. Although ruminal concentration of total volatile fatty acids (VFA) and the proportion of individual VFA were partially affected by administration of soy sauce oil, values were within normal ranges, showing no apparent inhibition in rumen fermentation. Administration of soy sauce oil decreased the proportions of milk fatty acids with a carbon chain length of less than 18, and increased the proportions of stearic, oleic, vaccenic and conjugated linoleic acids. Conjugated linoleic acid content in milk became 5.9-8.8 times higher with soy sauce oil administration. Blood serum concentrations of non-esterified fatty acid, 3-hydroxybutyric acid, total cholesterol, free cholesterol, esterified cholesterol, triglyceride and phospholipid increased with administration of soy sauce oil, suggesting a higher energy status of the experimental cows. Conclusion: The results suggest that soy sauce oil could be a useful supplement to potentially improve milk functionality without adverse effects on ruminal fermentation and animal health. More detailed analysis is surely necessary to optimize the supplementation level of this new lipid source in feeding trials.