Cdc50 is the non-catalytic subunit of the flippase that establishes phospholipid asymmetry in membranes and functions in vesicle-mediated trafficking in Saccharomyces cerevisiae. Here, we have identified the homologous gene CaCDC50 that encodes a protein of 396 amino acids with two conserved transmembrane domains in Candidaalbicans. Deletion of CaCDC50 results in C. albicans cells becoming sensitive to the antifungal drugs azoles, terbinafine and caspofungin, as well as to the membrane-perturbing agent sodium dodecyl sulfate. We also show that CaCDC50 is involved in both endocytosis and vacuolar function. CaCDC50 confers tolerance to high concentrations of cations, although it is not required for osmolar response. Moreover, deletion of CaCDC50 leads to severe defects in hyphal development of C. albicans cells and highly attenuated virulence in the mouse model of systemic infection. Therefore, CaCDC50 regulates cellular responses to antifungal drugs, cell membrane stress, endocytosis, filamentation and virulence in the human fungal pathogen C. albicans.