Repeated and prolonged use of fluconazole in treating candidosis leads to drug resistance. The aim of this study was to confirm the relationship between T916C (Y257H) mutation in Candida albicans ERG11 and fluconazole resistance. We replaced one copy of ERG11 with ERG11 containing the T916C mutation in C. albicans CAI4 and expressed ERG11 with the T916C mutation in Saccharomyces cerevisiae INVSc1. The MIC values were two- to four-fold greater in CAI4 transformants with than without the T916C mutation and 128 and 32 μg ml−1 for S. cerevisiae INVSc1-containing ERG11 with and without the T916C mutation. T916C mutation may be associated with fluconazole resistance in C. albicans.