Antifungals targeting membrane ergosterol are longstanding, yet indispensable drugs in clinical use. However, the mechanisms by which the cellular membrane domains recognized by these antibiotics are generated remain largely unknown. Here, we demonstrate that the balance between endocytosis and exocytosis in membrane trafficking is a critical factor in the action of sterol-targeting antibiotics. When fission yeast cells were treated with manumycin A, cellular binding and the action of the antifungals filipin, amphotericin B, and theonellamides, all of which are ergosterol-binders, were abolished. Additionally, manumycin A treatment attenuated Cdc42 activity and inhibited exocytosis, while endocytosis was only moderately suppressed. Similar defects in membrane trafficking could be reproduced by heat shock and genetic perturbation, which also abolished the action of the antibiotics. We propose that exocytosis and endocytosis respectively supply and internalize the specific plasma membrane domains recognized by sterol-targeting antibiotics.