Acyl CoA-monoacylglycerol acyltransferase (MGAT) catalyzes the first step in triacyglycerol resynthesis involved in dietary absorption in enterocytes. Despite its potentially important role in dietary fat absorption, a gene encoding a human intestinal MGAT has not been identified. In this study, we report the identification and functional characterization of a human intestinal MGAT (hMGAT2) and its splice variant (hMGAT2V). The hMGAT2 gene encodes a peptide of 334 amino acids with a molecular mass of 38.2 kDa that shares 81 and 47% amino acid identities with the mouse MGAT2 and the human diacylglycerol acyltransferase (DGAT2) enzymes, respectively. The hMGAT2 gene is localized on chromosome 11q13.5, adjacent to the DGAT2 gene, suggesting gene duplication. Transient expression of hMGAT2, but not an alternatively spliced variant, hMGAT2V, in COS-7 cells led to a ninefold increase in the synthesis of DAG. The human and mouse differ significantly in tissue distribution of MGAT2. In addition to a predominant expression in the small intestine in both species, distinct levels were also found in the human liver, contrasting with higher levels in the mouse kidney. In comparison with a single 1.8-kb transcript in mouse, the hMGAT2 gene expressed two transcripts of 3.0 and 6.0 kb in size that encode MGAT2 and an inactive peptide with unknown functions, respectively. Despite a significant level of hMGAT2 mRNA in the human liver, little MGAT activity was detected in liver microsomes when tested against monoacyglcerols with different unsaturated side chains, suggesting possible posttranscriptional regulation.