MCF-10A breast epithelial cells treated with docosahexaenoic acid (DHA) or oleic acid (OA) accumulated cytoplasmic lipid droplets containing both triacylglycerol and cholesteryl esters (CE). Interestingly, total CE mass was reduced in cells treated with DHA compared to cells treated with OA, and the CEs were rich in n-3 fatty acids. Thus, we hypothesized that DHA may be, in addition to a substrate, an inhibitor of cholesterol esterification in MCF-10A cells. We determined that the primary isoform of acyl-CoA: cholesterol acyltransferase expressed in MCF-10A cells is ACAT1. We investigated CE formation with DHA, OA, and the combination in intact cells and isolated microsomes. In both cells and microsomes, the rate of CE formation was faster and more CE was formed with OA compared to DHA. DHA substantially reduced CE formation when given in combination with OA. These data suggest for the first time that DHA can act as a substrate for ACAT1. In the manner of a poor substrate, DHA also inhibited the activity of ACAT1, a universally expressed enzyme involved in intracellular cholesterol homeostasis, in a cell type that does not secrete lipids or express ACAT2.