ABSTRACTInvasive aspergillosis (IA) is the second most common invasive fungal disease and is associated with high mortality rates.Aspergillus fumigatusis the predominant causal agent of this life-threatening infection. Triazoles are still the cornerstone of antifungal treatment, and voriconazole remains the first-line choice. However, voriconazole resistance has been increasingly reported, which results in significantly higher mortality rates for IA and is particularly problematic. In the present study, we report the identification and functional study of a protein with previously unknown function that is encoded by the gene designatedAfu-emi1(AFUA_1G07360). High-throughput gene replacement technology was applied to construct the knockout ΔAfu-emi1strain and a revertant strain. The MICs for azoles, including posaconazole, itraconazole, and voriconazole, were evaluated via the broth microdilution method and E-tests, which revealed that disruption ofAfu-emi1resulted in 4-fold increased susceptibility to voriconazole. Colony growth in the presence of oxidants, namely, H2O2and menadione, and osmotic pressure-altering agents, namely, NaCl andd-sorbitol, was measured. TheAfu-emi1mutant strain exhibited a significant growth defect under oxidative and osmotic stress. The reactive oxygen species (ROS) production levels with or without voriconazole pretreatment were determined, and theAfu-emi1mutant strain exhibited significantly lower ROS production levels. The effects ofAfu-emi1disruption on voriconazole susceptibility, growth under stress, and ROS production were restored in the revertant strain. In addition, the expression ofcyp51A,AfuMDR2,AfuMDR3,AfuMDR4, andcdr1bin the ΔAfu-emi1strain was significantly reduced. In conclusion, deletion of the geneAfu-emi1resulted in increased voriconazole susceptibility, attenuated ability for oxidative and osmotic stress adaptation, decreased ROS production, and downregulation ofcyp51A, AfuMDR2,AfuMDR3,AfuMDR4, andcdr1bexpression, suggesting that Afu-Emi1 is an important regulator of stress adaptation andcyp51Aand efflux pump expression in this medically important fungus.IMPORTANCEVoriconazole is the first-line choice for IA, a life-threatening disease. Therefore, voriconazole resistance has become particularly problematic. Disruption ofAfu-emi1resulted in increased susceptibility to voriconazole, a significant growth defect under oxidative and osmotic stress, and downregulation of target enzyme Cyp51A and efflux pump expression, suggesting that Afu-Emi1 is an important regulator of stress adaptation andcyp51Aand efflux pump expression in this medically important fungus. Targeting Afu-Emi1 might help to enhance azole therapeutic efficacy and impede azole resistance.