Mammary epithelial cells, which express and secrete leptin into milk, accumulate triacylglycerol (TAG). We examined effects on the accumulation of cytosolic TAG of addition of short- (acetate and butyrate) or medium- (octanoate) chain fatty acids to the medium bathing bovine mammary epithelial cells (bMEC). Octanoate stimulated the accumulation of TAG in a concentration-dependent manner from 1 to 10 mM and increased lipid droplet formation and mRNA expression of CD36 (a fatty acid translocase). Additionally, expression of a peroxisome proliferator activated receptor (PPAR) γ 2 protein that is a lipid-activated transcription factor, was increased by the addition of acetate or octanoate. However, leptin mRNA expression was significantly reduced by addition of acetate or butyrate. Both short- and medium-chain fatty acids inhibited acetyl coenzyme A carboxylase (ACC) activities, which is pivotal in lipid synthesis, but elevated expression of uncoupling protein 2 (UCP2) mRNA, which is important in energy expenditure. These results suggest that octanoate induces cytosolic TAG accumulation and the formation of lipid droplets, and that acetate and butyrate inhibit leptin expression and lipid synthesis in bMEC.