Background: Trehalose is a functional disaccharide that has anti-metabolic activities such as suppression of adipocyte hypertrophy in mice and alleviation of impaired glucose tolerance in humans. Trehalase hydrolyzes trehalose in the small intestine into two glucose molecules. In this study, we investigated whether trehalose can suppress adipocyte hypertrophy in mice in the presence or absence of trehalase. Methods: Trehalase knockout (KO) mice and wild-type (WT) mice were fed a high fat diet (HFD) and administered water with 0.3% (w/v) or without trehalose for 8 weeks. At the end of the experimental period, mesenteric adipose tissues and the small intestine were collected and the adipocyte size and proportion of cytoplasmic lipid droplets (CLDs, %) in jejunum epithelium were measured by image analysis. Results: Trehalose treatment was associated with suppressed adipocyte hypertrophy in both trehalase KO and WT mice. The rate of CLDs in the jejunal epithelium was increased in both trehalase KO and WT mice given water containing trehalose relative to untreated control mice. Since there was a negative correlation between jejunal epithelial lipid droplet volume and mesenteric adipocyte size, together with these results, trehalose treatment would suppress adipocyte hypertrophy. Because of jejunal epithelium containing lipid droplets falled into the intestinal lumen, triglyceride (TG) levels in feces tended to be higher in the KO/HFD/Tre group than in the KO/HFD/Water group. Whereas feces from trehalose-treated trehalase KO and WT mice tended to have more free fatty acids (FFA) than the untreated groups. Chylomicron-TG tended to be decreased in both trehalose-treated trehalase KO and WT mice. In vitro , addition of trehalose to differentiated Caco-2 cells increased intracytoplasmic lipid droplets and decreased secretion of the chylomicron marker ApoB48. Conclusions: The suppression of adipocyte hypertrophy in the presence and absence of trehalase indicates that trehalose mediates effects prior to being hydrolyzed into glucose. In both trehalase KO and WT mice, trehalose treatment increased the rate of CLDs in jejunal epithelium, reduced chylomicron migration from the intestinal epithelium to the periphery, and suppressed adipocyte hypertrophy. Thus, trehalose ingestion could prevent metabolic syndrome by trapping fat droplets in the intestinal epithelium and suppressing rapid increases in chylomicrons.