Candida albicans cells homozygous at the mating-type locus stochastically undergo the white-to-opaque switching to become mating-competent. This switching is regulated by a core circuit of transcription factors organized through interlocking feedback loops around the master regulator Wor1. Although a range of distinct environmental cues is known to induce the switching, the pathways linking the external stimuli to the central control mechanism remains largely unknown. By screening a C. albicans haploid gene-deletion library, we found that SAC7 encoding a GTPase-activating protein of Rho1 is required for the white-to-opaque switching. We demonstrate that Sac7 physically associates with Rho1-GTP and the constitutively active Rho1G18V mutant impairs the white-to-opaque switching while the inactive Rho1D124A mutant promotes it. Overexpressing WOR1 in both sac7Δ/Δ and rho1 G18V cells suppresses the switching defect, indicating that the Sac7/Rho1 module acts upstream of Wor1. Furthermore, we provide evidence that Sac7/Rho1 functions in a pathway independent of the Ras/cAMP pathway which has previously been positioned upstream of Wor1. Taken together, we have discovered new regulators and a signaling pathway that regulate the white-to-opaque switching in the most prevalent human fungal pathogen C. albicans.