Amphotericin B (AmB) is an antifungal antibiotic which, despite the severe side effects, is still used for the treatment of systemic fungal infections. Herein we studied the influence of pH upon the selectivity and the transport properties of AmB channels inserted in reconstituted, ergosterol-containing zwitterionic lipid membranes. Our electrophysiology experiments carried out on single and multiple AmB channels prove that at pH 2.8 these channels are anion selective, whereas at neutral and alkaline pH's (pH 7 and pH 11) they become cation selective. We attribute this to the pH-dependent ionization state of the carboxyl and amino groups present at the mouth of AmB molecules. Surprisingly, our data reveal that the single-molecule ionic conductance of AmB channels varies in a non-monotonic fashion with pH changes, which we attribute to the pH-dependent variation of the surface and dipole membrane potential. We demonstrate that when added only from one side of the membrane, in symmetrical salt solutions across the membrane and low pH values, AmB channels display a strong rectifying behavior, and their insertion is strongly favored when positive potentials are present on the side of their addition.