Antimicrobial activities of many defense proteins are profoundly altered by inorganic cations, thereby controlling disease pathologies in a number of mammalian systems, such as cystic fibrosis in humans. Protein-based active defense systems in plants also are influenced by cations; however, little is known of how these cation effects are mediated. Cytotoxicity of the pathogenesis-related protein osmotin against the model fungus Saccharomyces cerevisiae was progressively abolished by K+. By the use of S. cerevisiae mannosylation mutants, this effect was shown to require mannosephosphate residues in the cell wall. However, osmotin activity was not suppressed by even high concentrations of Ca2+. Rather, submillimolar levels of Ca2+ specifically facilitated osmotin's activity, as well as its binding to the cell surface. This effect also was dependent on mannosephosphate groups on the cell surface, and appeared to require negative charge on a portion of the osmotin protein. Results suggest that Ca2+ modulates osmotin action by facilitating its binding to the fungal cell surface, but that K+ blocks this interaction by competing for binding to mannosephosphate groups. Therefore, we have identified glycan interaction as a mechanism for antimicrobial protein activity modulation by cations, a pattern that may apply to diverse innate defense responses.