Trichophyton rubrum is the predominant causative agent for superficial dermatomycosis. In order to understand how triazole antifungal agents interact with dermatophytes, the gene expression response of T. rubrum to itraconazole was studied by large-scale gene expression profiling. A total of 670 genes were found to be responsive to itraconazole, including 305 that were up-regulated and 365 down-regulated. Most genes involved in lipid metabolism and especially in ergosterol biosynthesis were up-regulated in response to itraconazole, including ERG6, ERG7, ERG11, ERG24, ERG25 and ERG26. In addition, transcription of some genes involved in cell stress response, drug efflux, and small molecule transport was also affected by itraconazole. Differential expression of selected genes was confirmed by real-time reverse transcription polymerase chain reaction (RT-PCR). This is the first microarray hybridization analysis of T. rubrum exposed to a triazole antifungal agent.