Amphitropic proteins, such as the virulence factor phosphatidylinositol-specific phospholipase C (PI-PLC) from Bacillus thuringiensis, often depend on lipid-specific recognition of target membranes. However, the recognition mechanisms for zwitterionic lipids, such as phosphatidylcholine, which is enriched in the outer leaflet of eukaryotic cells, are not well understood. A 500 ns long molecular dynamics simulation of PI-PLC at the surface of a lipid bilayer revealed a strikingly high number of interactions between tyrosines at the interfacial binding site and lipid choline groups with structures characteristic of cation-pi interactions. Membrane affinities of PI-PLC tyrosine variants mostly tracked the simulation results, falling into two classes: (i) those with minor losses in affinity, K-d(mutant)/K-d(wild-type) <= 5 and (ii) those where the apparent K-d was 50-200 times higher than wild-type. Estimating Delta Delta G for these Tyr/PC interactions from the apparent K-d values reveals that the free energy associated with class I is similar to 1 kcal/mol, comparable to the value predicted by the Wimley-White hydrophobicity scale. In contrast, removal of class II tyrosines has a higher energy cost: similar to 2.5 kcal/mol toward pure PC vesicles. These higher energies correlate well with the occupancy of the cation-pi adducts throughout the MD simulation. Together, these results strongly indicate that PI-PLC interacts with PC headgroups via cation-pi interactions with tyrosine residues and suggest that cation-pi interactions at the interface may be a mechanism for specific lipid recognition by amphitropic and membrane proteins.