Histatin-resistant derivatives of Candida albicans strain 132A, generated by successive exposure to increasing concentrations of histatin 3, were previously reported to be similar to the parent strain in their histatin binding, internalization, oxygen consumption, ATP efflux, and histatin degradation. Proteomic analysis of further histatin-resistant secondary derivatives of this series revealed that 59 proteins were differentially expressed compared to the parental strain. Of these 59 proteins, 3 were absent in histatin-resistant secondary derivatives and 11 were absent in the parent strain. Of the proteins absent in the histatin-resistant derivatives, the most notable was elongation factor 2, a target for the natural antifungal sordarin. Of the proteins absent in the parent strain but present in histatin-resistant derivatives, those identified included isocitrate lyase (Icl1p), fructose biphosphate aldolase (Fba1p), pyruvate decarboxylase (Pdc2p), and ketol-acid reductoisomerase (Ilv5p). The present secondary derivatives showed significantly decreased rates of oxygen consumption and histatin 3-mediated ATP release compared to the parent strain and also showed stability of the histatin-resistant phenotype. A significant (twofold) decrease in transcript levels of the potassium transporter encoded by TRK1, a critical mediator of histatin killing, was found in only one of the secondary histatin-resistant derivatives compared to the parent strain. The sequential exposure of C. albicans to histatin 3 described here resulted in the induction or selection of a phenotype with impaired metabolic function. The results support an important role for metabolic pathways in the histatin resistance mechanism and suggest that there may be several intracellular targets for histatin 3 in C. albicans.