This study investigated the effect of a single oral ingestion of alpha-linolenic acid-enriched diacylglycerol (ALA-DAG) on postprandial serum triglyceride (TG) levels. A randomized, double-blind, controlled, crossover study was performed in subjects with normal or moderately high fasting serum TG levels. Subjects ingested 0.00 g [control: triacylglycerol; TAG (rapeseed oil)], 1.25 g (1.25-g: mixture of 1.25 g ALA-DAG and 1.25 g TAG), or 2.50 g (2.50 g) of ALA-DAG in random order with a 6-d washout period. Serum TG levels were evaluated in the fasting state, and at 2, 3, 4, and 6 h after the test meal. Thirty-eight subjects completed the study and were defined as the per protocol set. As the primary outcome, postprandial serum TG levels were significantly lower in the 2.50-g treatment compared with the control. The TG level did not differ significantly between the 1.25-g and control. The suppressive effect of ALA-DAG on the serum TG level correlated significantly with the body mass index and fasting insulin level. ALA-DAG at a dose of 2.50 g had greater effects on serum TG and apolipoprotein B levels in subjects with a higher body mass index (≥25 kg/m2) and higher fasting serum insulin levels (>10 μU/mL). Our findings suggest that ingesting 2.50 g ALA-DAG suppresses the postprandial serum TG level in people with normal and moderately high fasting serum TG levels, presumably as a result of poor re-esterification of dietary fat into TG in the intestinal mucosa.