is a foodborne bacterial pathogen that causes listeriosis. Positive regulatory factor A (PrfA) is a pleiotropic master activator of virulence genes of that becomes active upon the entry of the bacterium into the cytosol of infected cells. can survive and multiply at low temperatures; this is accomplished through the maintenance of appropriate membrane fluidity via branched-chain fatty acid (BCFA) synthesis. Branched-chain α-keto acid dehydrogenase (BKD), which is composed of four polypeptides encoded by , , , and , is known to play a vital role in BCFA biosynthesis. Here, we constructed BKD-deficient strains by in-frame deletion of , , , and genes. To determine the role in and , mouse model challenges, plaque assay in murine L2 fibroblast, and intracellular replication in J744A.1 macrophage were conducted. BKD-deficient strains exhibited defects in BCFA composition, virulence, and PrfA-regulon function within the host cells. Transcriptomics analysis revealed that the transcript level of the PrfA-regulon was lower in Δ strain than those in the wild-type. This study demonstrates that strains lacking BKD complex components were defective in PrfA-regulon function, and full activation of wild-type may not occur within host cells in the absence of BKD. Further study will investigate the consequences of BKD deletion on PrfA function through altering BCFA catabolism.IMPORTANCE is the causative agent of listeriosis, a disease with a high mortality rate. In this study, we have shown that the deletion of BKD can impact the function of PrfA and the PrfA-regulon. The production of virulence proteins within host cells is necessary for to promote its intracellular survival and is likely dependent on membrane integrity. We thus report a link between membrane integrity and the function of PrfA. This knowledge will increase our understanding of pathogenesis, which may provide insight into the development of antimicrobial agents.