The roles of Candida albicans CHK1, a key gene from two-component system, in oral mucosal infection are not clear. This study evaluated the key roles of CHK1 gene in vitro and in vivo. The expression of CHK1 and its regulated virulence factors were tested during the oral epithelial cell infection. The production of lactate dehydrogenase, ROS, and IL-1 alpha combined with the confocal and scanning electron microscope observation was employed to identify the capability of CHK1 in damaging the epithelial cells. Both immunocompetent and immunodeficient mice oropharyngeal infection models were involved to confirm the roles of CHK1 gene in vivo. The expression of CHK1 gene was significantly increased during the oral epithelial cell infection. The chk1 Delta/Delta mutant failed to damage the epithelial cells or induce IL-alpha and ROS production. Interestingly, chk1 Delta/Delta can also form the similar hyphae with WT and complementary strains. Accordingly, chk1 Delta/Delta did not affect the adhesion and invasion rates of C. albicans to oral epithelial cells. However, chk1 Delta/Delta significantly decreased the expression levels of the virulence factors, including ALS2, SAP6, and YWP1. The chk1 Delta/Delta also failed to cause oral candidiasis in both immunocompetent and immunodeficient mice indicating that CHK1 gene from the two-component system is essential for the pathogenicity of C. albicans.