Edited by James Rothman N-ethylmaleimide-sensitive fusion factor (NSF) is an ATPase that plays a crucial role in vesicular transport. Here, we examined the effects of NSF knockdown on Golgi structure and different vesicle trafficking pathways in mammalian cells. NSF knockdown caused Golgi fragmentation and abolished transferrin receptor exocytosis, defects that were rescued by RNAi-resistant NSF. Strikingly, NSF deficiency in HeLa cells barely affected cell viability, anterograde trafficking of vesicular stomatitis virus glycoprotein G and transferrin endocytosis. These results confirm the central role of NSF in Golgi structure and reveal differential requirement of NSF for exocytic recycling and constitutive trafficking pathways.