Invasive aspergillosis remains a life-threatening infection in immunocompromised patients. Although clinical failures are attributed to poor host immunity, antifungal drug resistance may be a contributing factor. Reports of voriconazole (VRC) resistance (VRC-R) in clinical isolates of Aspergillus spp. continue to emerge from various centers around the world, and mechanisms contributing to drug resistance are poorly understood. The aim of this study is to study the role of multidrug resistance efflux pumps (MDR-EPs) in VRC-R in Aspergillus flavus using efflux pump inhibitors and quantitative reverse transcriptase polymerase chain reaction. Relative quantification of various MDR-EPs was performed pre-exposure and postexposure to VRC, which demonstrated an increase in 1 or more efflux pump gene transcripts to varying degrees in VRC-susceptible and VRC-R isolates of A. flavus. Exposure to sub-MIC of VRC causes up-regulation of genes encoding MDR-EPs, contributing to triazole resistance in A. flavus and may not be detected during routine antifungal susceptibility testing in vitro.