Objective: The objective of this study was to examine the effect of dietary diacylglycerol (DAG) on the bioavailability of fat-soluble vitamins in comparison with triacylglycerol (TAG). Methods: We conducted a long-term administration test of DAG and TAG in 27 healthy men aged 27–47 years. After measuring baseline values, subjects were randomized into two groups, one group (n = 15) was given DAG and the other (n = 12) was given TAG. Subjects ingested 20 g of DAG or TAG either in mayonnaise or an emulsion drink of their own choice at meals once a day for 12 weeks. At 4, 8 and 12 weeks, fasting blood samples were drawn and serum levels of vitamin A, E, and D were measured. Results: There were no significant changes in vitamin A levels throughout the study period. Compared to the initial values (using a Student’s t test for paired values), significant differences of vitamin E and D were seen at some points during the experiment. According to a two-way repeated measures analysis of variance, however, DAG and TAG (lipid) and time had no effect on fat-soluble vitamin levels. Conclusions: Our results indicate that DAG does not affect the absorption of the fat-soluble vitamins in diets.