Acyl-CoA:monoacylglycerol transferase (MGAT) plays a predominant role in dietary fat absorption in the small intestine, where it catalyzes the first step of triacylglycerol resynthesis in enterocytes for chylomicron formation and secretion. Although the mouse small intestine exhibits the highest MGAT enzyme activity among all of the tissues studied, the gene encoding the enzyme has not been identified so far. In the present studies, we report the identification and characterization of a mouse intestinal MGAT, MGAT2. Transient expression of MGAT2 in AV-12, COS-7, and Caco-2 cells led to a more than 70-, 30-, and 35-fold increase in the synthesis of diacylglycerol, respectively. MGAT2 expressed in mammalian cells can catalyze the acylation of rac-1-, sn-2-, and sn-3-monoacylglycerols, and the enzyme prefers monoacylglycerols containing unsaturated fatty acyls as substrates. MGAT2 also demonstrates weak DGAT activity, which can be distinguished from its MGAT activity by detergent treatment that abolishes DGAT but not MGAT activity. We also analyzed the biochemical features of MGAT2 and demonstrated homogenate protein-, time-, and substrate concentration-dependent MGAT enzyme activity in transiently transfected COS-7 cells. Northern blot analysis indicates that the mouse MGAT2 is most abundantly expressed in the small intestine, suggesting that MGAT2 may play an important role in dietary fat absorption.