Benzalkonium chloride (BAC) is an effective disinfectant against Salmonella and widely used in the food industry. However, there are growing concerns about the risk of inducing Salmonella to produce cross-resistance to antibiotics resulting from the excessive use of BAC. In this study, 50 foodborne Salmonella isolates were exposed to sub-inhibitory concentrations of BAC. It was demonstrated that 7 isolates showed direct resistance to BAC, and 35 isolates showed cross-resistance to at least one of 10 tested antibiotics. Among these 35 isolates, 15 isolates exhibited an increase in minimum inhibitory concentration (MIC) for tetracycline, followed by 11 isolates for kanamycin and 9 isolates for ampicillin. Salmonella Enteritidis SJTUSM06 with the highest increase in MIC of tetracycline (from 4 to 32 μg/mL) was selected for further study. Tandem mass tag-labeled proteomics was used to determine key proteins involved in development of cross-resistance to tetracycline in BAC-adapted S. Enteritidis. It was identified that there were 146 differentially expressed proteins, including 95 up-regulated and 51 down-regulated proteins, most of which were involved in carbohydrate and lipid metabolism, ribosomes, transporters, virulence, motility, and stress response pathways. The efflux pump AcrB was significantly upregulated by 2.03-fold in BAC-adapted S. Enteritidis. It was also demonstrated that efflux pump inhibitor phenylalanine-arginine β-naphthylamide (PaβN) decreased the MIC of tetracycline in BAC-adapted S. Enteritidis from 32 to 8 μg/mL, indicating that active efflux pump played an important role in the development of resistance to tetracycline induced by BAC. Deletion of acrB resulted in a decrease in the resistance to tetracycline in BAC-adapted S. Enteritidis. Compared with the parent strain, MIC for tetracycline in ΔacrB mutant was 16 μg/mL with a 2-fold decrease. These results demonstrated that AcrB played a positive role in the development of cross-resistance to tetracycline after adaptation of S. Enteritidis to BAC.