Angiopoietin-like protein 3 (ANGPTL3) is an important inhibitor of lipoprotein lipase and endothelial lipase that plays critical roles in lipoprotein metabolism. It specifically expresses in the liver and undergoes proprotein convertase-mediated cleavage during secretion, which generates an N-terminal coiled-coil domain and C-terminal fibrinogen-like domain that has been considered as the activation step for its function. Previous studies have reported that the polypeptide GalNAc-transferase GALNT2 mediates the O-glycosylation of the ANGPTL3 near the cleavage site, which inhibits the proprotein convertase (PC)-mediated cleavage in vitro and in cultured cells. However, loss-of-function mutation for GALNT2 has no effect on ANGPTL3 cleavage in human. Thus whether GALNT2 regulates the cleavage of ANGPTL3 in vivo is unclear. In present study, we systematically characterized the cleavage of Angptl3 in cultured cells and in vivo of mice. We found that endogenous Angptl3 is cleaved in primary hepatocytes and in vivo of mice, and this cleavage can be blocked by Galnt2 overexpression or PC inhibition. Moreover, suppressing galnt2 expression increases the cleavage of Angptl3 in mice dramatically. Thus, our results support the conclusion that Galnt2 is a key endogenous regulator for Angptl3 cleavage both in vitro and in vivo.