Background: Monoacylglycerol acyltransferase 2 (MGAT2) is an enzyme involved in triglyceride resynthesis of small intestinal epithelial cells. Although some reports indicate that inhibition of MGAT2 would be a promising target for anti-obesity treatment, there are few MGAT2 inhibitors that show a highly effective profile. Therefore, we identified S-309309, which is an oral MGAT2 small molecule inhibitor, and examined its anti-obesity effect and mechanisms in non-clinical study. Methods: In vitro activity of S-309309 was tested using mouse and human MGATs and diacylglycerol acyltransferase (DGATs) enzymes. In vivo anti-obesity effect of S-309309 was evaluated in high fat diet induced obese (DIO) mice by comparison with liraglutide or combination of phentermine/topiramate, which used in clinical for obesity. In addition, combination studies with liraglutide or semaglutide and mechanism study of S-309309 anti-obesity effect were also conducted in DIO mice. Results: S-309309 had potent inhibitory activity for human and mouse MGAT2 with IC50 values of <30 nM. On the other hand, IC50 values of S-309309 on human MGAT3, DGAT1 and DGAT2 were more than 100 pM. S-309309 showed a potent and dose-dependent anti-obesity effect in DIO mice, and the effect was stronger than that of liraglutide and combination of phentermine/topiramate. When combination with liraglutide or semaglutide, the combinations exhibited stronger anti-obesity effect than single-agent therapy of liraglutide, semaglutide or S-309309 in DIO mice. Furthermore, we demonstrated that S-309309 had multiple mechanisms of action such as suppression of feeding, delayed lipid absorption into the body and increased energy expenditure. Conclusions: S-309309 showed a significant and selective inhibitory activity on human and mouse MGAT2 in vitro and a potent antiobesity effect with multiple mechanisms in DIO mice. These results suggest the attractive therapeutic potential of S-309309 for obesity treatment.