Amphotericin B is the archetype for small molecules that form transmembrane ion channels. However, despite extensive study for more than five decades, even the most basic features of this channel structure and its contributions to the antifungal activities of this natural product have remained unclear. We herein report that a powerful series of functional group-deficient probes have revealed many key underpinnings of the ion channel and antifungal activities of amphotericin B. Specifically, in stark contrast to two leading models, polar interactions between mycosamine and carboxylic acid appendages on neighboring amphotericin B molecules are not required for ion channel formation, nor are these functional groups required for binding to phospholipid bilayers. Alternatively, consistent with a previously unconfirmed third hypothesis, the mycosamine sugar is strictly required for promoting a direct binding interaction between amphotericin B and ergosterol. The same is true for cholesterol. Synthetically deleting this appendage also completely abolishes ion channel and antifungal activities. All of these results are consistent with the conclusion that a mycosamine-mediated direct binding interaction between amphotericin B and ergosterol is required for both forming ion channels and killing yeast cells. The enhanced understanding of amphotericin B function derived from these synthesis-enabled studies has helped set the stage for the more effective harnessing of the remarkable ion channel-forming capacity of this prototypical small molecule natural product.