ABSTRACTThe yeast-to-hyphal morphotype transition and subsequent biofilm formation are important virulence factors ofCandida albicansand are closely associated with ergosterol biosynthesis. Flo8 is an important transcription factor that determines filamentous growth and biofilm formation inC. albicans. However, the relationship between Flo8 and regulation of the ergosterol biosynthesis pathway remains elusive. Here, we analyzed the sterol composition of aflo8-deficientC. albicansstrain by gas chromatography-mass spectrometry and observed the accumulation of the sterol intermediate zymosterol, the substrate of Erg6 (C-24 sterol methyltransferase). Accordingly, the transcription level ofERG6was reduced in theflo8-deficient strain. Yeast one-hybrid experiments revealed that Flo8 physically interacted with theERG6promoter. Ectopic overexpression ofERG6in theflo8-deficient strain partially restored biofilm formation andin vivovirulence in aGalleria mellonellainfection model. These findings suggest that Erg6 is a downstream effector of the transcription factor Flo8 that mediates the cross talk between sterol synthesis and virulence factors inC. albicans.IMPORTANCEBiofilm formation byC. albicanshinders its eradication by immune cells and antifungal drugs. Flo8 is an important morphogenetic transcription factor that regulates the biofilm formation andin vivovirulence ofC. albicans. However, little is known about how Flo8 regulates biofilm formation and fungal pathogenicity. Here, we determined that Flo8 directly binds to the promoter ofERG6to positively regulate its transcriptional expression. Consistently, loss offlo8results in the accumulation of the substrate of Erg6. Moreover, ectopic overexpression ofERG6at least partially restores the biofilm formation and virulence of theflo8-deficient strain bothin vitroandin vivo. This work provides a new perspective on the metabolic link between transcription factors and morphotypes inC. albicans.