Amphotericin B (AmB) is one of the most efficient antimycotic drugs used in clinical practice. AmB interacts with membrane sterols increasing permeability of fungal membranes; however, it is still unclear how AmB selectively recognizes the fungal sterol, ergosterol, over other sterols in cell membranes. In this study, we investigated the effect of an ergosterol side chain on AmB activity by testing a series of ergosterol analogues that shared the same alicyclic structure as ergosterol but varied in the side chain structure by using the K+ influx assay. The results clearly showed that the sterol side chain is essential for AmB selectivity towards ergosterol and for the activity of AmB-sterol ion channels. In agreement with our previous findings showing the direct interaction between the drug and ergosterol, these data suggested that AmB directly recognizes the sterol side chain structure, consequently promoting the formation of ion channels by AmB. Furthermore, the C24 methyl group and Δ22 double bond in the ergosterol side chain are equally important for the interaction with AmB. Conformational analysis revealed that the C24 methyl group contributes to the interaction by increasing the van der Waals (VDW) contact area of the side chain, while the Δ22 double bond restricts the side chain conformation to maximize the VDW contact with the rigid AmB aglycone. This study provides direct experimental evidence of the mechanism of AmB selectivity towards fungal ergosterol.