Mitochondrial porins (also known as voltage-dependent anion-selective channels (VDACs)) regulate and contribute to cellular metabolism. These proteins copurify with sterols, and some purified forms of the protein require sterol for insertion into planar artificial membranes. Recently, interactions between detergent-solubilized mitochondrial porins and sterols have been detected by NMR and spectroscopic methods, but the effects of sterols on pore function remained to be assessed. Therefore, in this work, a freeze-thaw technique was used to introduce recombinant Neurospora porin into liposomes containing, or lacking, the native fungal sterol ergosterol. In both types of liposomes, insertion of the protein converts it to a protease-resistant state and low levels of dimeric and trimeric forms are observed. There are only minor differences between the secondary structural components of the protein in the presence or absence of sterol. Ergosterol in proteoliposomes alters their osmotic responses to sucrose, possibly due to increased membrane rigidity or interactions with the protein that were not revealed by the methods used in this study. The presence of ergosterol is associated with an increased change in conformation and loss of function of liposome-embedded porin at high temperature. Taken with other evidence for direct interactions of sterols with porins, these results support a link between these two molecules in mitochondrial membrane activity.