Benzalkonium chloride (BC) is a well-known quaternary ammonium compound (QAC) that has been widely used as surface disinfectants in the food processing. To date, little is known about the underlying mechanisms of how bacterial pathogens respond to QACs and develop resistance against them. In this study, the genome-wide transcriptional responses of Salmonella Typhimurium exposed to BC were investigated. Interestingly, Salmonella pathogenicity island 1 (SPI-1) genes were down-regulated under sublethal condition of BC. In the down-regulation of SPI-1 genes by BC treatments, HilD, a master regulator of SPI-1, was found to be decreased. However, the absence of Lon protease, which degrades HilD, nearly nullified the BC-mediated repression of SPI-1 genes. This result implies that BC represses SPI-1 expression by modulating Lon activity. Furthermore, the expression of ramA was strongly induced by BC treatments and the deletion of ramA rendered Salmonella susceptible to BC treatments, suggesting the positive role of RamA in BC tolerance. RamA is known to play a role as a transcriptional activator for acrAB-tolC encoding multidrug efflux system. The attenuated BC resistance of ΔramA mutant strain was complemented by the introduction of AcrA in trans, indicating that AcrAB-TolC efflux system is required for Salmonella resistance to BC. Besides, introduction of RamA repressed the transcription of SPI-1 genes, however, its negative role in SPI-1 expression was likely to be independent of the Lon-mediated regulation of SPI-1. These results demonstrated that sublethal BC treatments not only stimulated Salmonella to develop resistance mechanisms against BC but also influenced Salmonella virulence. This study provided new insights into the comprehensive transcriptional responses of Salmonella to BC.