Candida glabrata is an opportunistic pathogen with increasing clinical significance due to its innate antifungal resistance. This study reveals the critical role of the phospholipid flippase Drs2 (P4-ATPase) in maintaining fungal pathogenicity and stress adaptation. The DRS2 deletion mutant exhibited impaired growth, heightened drug susceptibility, and defective stress responses, including compromised oxidative stress tolerance and autophagy. Importantly, the mutant showed reduced survival in macrophages while eliciting stronger proinflammatory responses, along with attenuated virulence in a Galleria mellonella infection model. Further analysis demonstrated diminished biofilm formation and impaired metabolic flexibility under nutrient-limited conditions. Through transcriptomic profiling, we identified Drs2 as a key regulator of MAPK signaling pathways, cell wall integrity, and oxidative stress responses. These findings collectively establish Drs2 as a central coordinator of membrane homeostasis that critically links cellular stress adaptation to virulence in C. glabrata. These findings identify Drs2 as a promising target for antifungal intervention and provide new insight into the biological functions of lipid flippases in fungal pathogens.