Acyl CoA:monoacylglycerol acyltransferase (MGAT) catalyzes the first step in triacyglycerol re-synthesis involved in dietary absorption in enterocytes. Despite its potential 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 MW of 38.2 kDa that shares 81% and 47% amino acid identities with the mouse MGAT2 and the human 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 9-fold increase in the synthesis of diacylglycerols. 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 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 encodes 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 post-transcriptional regulation.