AbstractEnniatins are mycotoxins with well‐known antibacterial, antifungal, antihelmintic and antiviral activity, which have recently come to attention as potential mitochondriotoxic anticancer agents. The cytotoxicity of enniatins is traced back to ionophoric properties, in which the cyclodepsipeptidic structure results in enniatin:cation‐complexes of various stoichiometries proposed as membrane‐active species. In this work, we employed a combination of surface‐enhanced infrared absorption (SEIRA) spectroscopy, tethered bilayer lipid membranes (tBLMs) and density functional theory (DFT)‐based computational spectroscopy to monitor the cation‐dependence (Mz+=Na+, K+, Cs+, Li+, Mg2+, Ca2+) on the mechanism of enniatin B (EB) incorporation into membranes and identify the functionally relevant EBn : Mz+ complexes formed. We find that Na+ promotes a cooperative incorporation, modelled via an autocatalytic mechanism and mediated by a distorted 2 : 1‐EB2 : Na+ complex. K+ (and Cs+) leads to a direct but less efficient insertion into membranes due to the adoption of “ideal” EB2 : K+ sandwich complexes. In contrast, the presence of Li+, Mg2+, and Ca2+ causes a (partial) extraction of EB from the membrane via the formation of “belted” 1 : 1‐EB : Mz+ complexes, which screen the cationic charge less efficiently. Our results point to a relevance of the cation dependence for the transport into the malignant cells where the mitochondriotoxic anticancer activity is exerted.