Non-alcoholic liver disease (NAFLD) is a condition caused by excessive fat accumulation in the liver and developed via multiple pathways.miR-27bhas been suggested to play crucial roles in the development of NAFLD, assuming via targeting genes involved in lipid catabolism and anabolism. However, other pathways regulated bymiR-27bare largely unknown. Here we show that lipid accumulation was induced inmiR-27b–transfectedhumanandmousehepatic cells and that knockdowns of three miR-27b–target genes,β-1,4-galactosyltransferase 3(B4GALT3),matrix AAA peptidase interacting protein 1(MAIP1) andPH domain and leucine rich repeat protein phosphatase 2(PHLPP2), induced lipid accumulation. We also show thatB4GALT3andMAIP1were direct targets ofmiR-27band overexpression ofMAIP1amelioratedmiR-27b−induced lipid accumulation. In addition, we show that hepaticMaip1expression declined in mice fed a high-fat diet, suggesting the involvement of decreasedMaip1expression in the condition of fatty liver. Overall, we identifiedMAIP1/miR-27baxis as a mediator of hepatic lipid accumulation, a potential therapeutic target for NAFLD.