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ABSTRACT Aspergillus fumigatus is the predominant pathogen responsible for aspergillosis infections, with emerging drug-resistant strains complicating treatment strategies. The role of mitochondrial functionality in fungal resistance to antifungal agents is well-documented yet not fully understood. In this study, the mitochondrial protein Bcs1A, a homolog of yeast Bcs1, was found to regulate colony growth, ion homeostasis, and the response to antifungal drugs in A. fumigatus. Microscopic observations revealed substantial colocalization of Bcs1A-GFP fusion protein fluorescence with mitochondria. Bcs1A deletion compromised colony growth and the utilization of non-fermentable carbon sources, alongside causing abnormal mitochondrial membrane potential and reduced reactive oxygen species production. These findings underscore Bcs1A’s vital role in maintaining mitochondrial integrity. Phenotypic analysis and determinations of minimum inhibitory concentrations indicated that the Δbcs1A mutant was more resistant to various antifungal agents, such as azoles, terbinafine, and simvastatin, compared to wild-type strain. RNA sequencing and RT-qPCR analysis highlighted an upregulation of multiple efflux pumps in the Δbcs1A mutant. Furthermore, loss of the principal drug efflux pump, mdr1, decreased azole tolerance in the Δbcs1A mutant, suggesting that Bcs1A’s modulated of azoles response via efflux pump expression. Collectively, these results establish Bcs1A as essential for growth and antifungal drug responsiveness in A. fumigatus mediated through mitochondrial regulation.IMPORTANCEDrug resistance presents a formidable obstacle in the clinical management of aspergillosis. Mitochondria are integral to various biochemical pathways, including those involved in fungi drug response, making mitochondrial proteins promising therapeutic targets for drug therapy. This study confirms that Bcs1A, a mitochondrial respiratory chain protein, is indispensable for mitochondrial functionality and multidrug tolerance in Aspergillus fumigatus. Mutation of Bcs1A not only leads to a series of drug efflux pumps upregulated but also shows that loss of the primary efflux pump, mdr1, partial reduction in drug tolerance in the Bcs1A mutant, highlighting that Bcs1A’s significant influence on mitochondria-mediated drug resistance.