Targeted gene deletion is now available for molecular genetic research of dermatophytes, and the physiological roles of several genes have been elucidated. However, this method cannot be applied to essential genes, which can be potential drug targets. To overcome this limitation, we have developed a conditional gene knockdown system using a copper-responsive promoter. The promoter sequence of the copper transporter gene CTR4 (P(CTR4)) and that of the copper efflux pump gene CRP1 (P(CRP1)) derived from Trichophyton rubrum were examined for their response to copper in Arthroderma vanbreuseghemii. P(CTR4) was demonstrated to repress expression of a reporter gene in the presence of copper, while the activity of P(CRP1) was induced by addition of copper. Importantly, P(CTR4) regulated the gene expression more tightly. Furthermore, when P(CTR4) was applied to regulate the expression of the endogenous genes ERG1 and TRP5, their conditional mutants exhibited decreased growth activity under the repressive conditions. These results suggest that the P(CTR4)-based gene regulation system represents a powerful tool for identification and characterization of a broad range of genes, including essential genes, in dermatophytes.