Many Candida albicans azole-resistant (AR) clinical isolates overexpress the CDR1 and CDR2 genes encoding homologous multidrug transporters of the ATP-binding cassette family. We show here that these strains also overexpress the PDR16 gene, the orthologue of Saccharomyces cerevisiae PDR16 encoding a phosphatidylinositol transfer protein of the Sec14p family. It has been reported that S. cerevisiae pdr16Delta mutants are hypersusceptible to azoles, suggesting that C. albicans PDR16 may contribute to azole resistance in these isolates. To address this question, we deleted both alleles of PDR16 in an AR clinical strain overexpressing the three genes, using the mycophenolic acid resistance flipper strategy. Our results show that the homozygous pdr16Delta/pdr16Delta mutant is approximately twofold less resistant to azoles than the parental strain whereas reintroducing a copy of PDR16 in the mutant restored azole resistance, demonstrating that this gene contributes to the AR phenotype of the cells. In addition, overexpression of PDR16 in azole-susceptible (AS) C. albicans and S. cerevisiae strains increased azole resistance by about twofold, indicating that an increased dosage of Pdr16p can confer low levels of azole resistance in the absence of additional molecular alterations. Taken together, these results demonstrate that PDR16 plays a role in C. albicans azole resistance.