The two-component system (TCS) is one of the primary pathways by which bacteria adapt to environmental stresses such as antibiotics. This study aimed to systematically explore the role of TCSs in the development of multidrug resistance (MDR) in Salmonella enterica serovar Enteritidis. Twenty-six in-frame deletion mutants of TCSs were generated from . Enteritidis SJTUF12367 (the wild type [WT]). Antimicrobial susceptibility tests with these mutants revealed that 10 TCSs were involved in the development of antibiotic resistance in . Enteritidis. In these 10 pairs of TCSs, functional defects in CpxAR, PhoPQ, and GlnGL in various . Enteritidis isolates led to a frequent decrease in MIC values against at least three classes of clinically important antibiotics, including cephalosporins and quinolones, which indicated the importance of these TCSs to the formation of MDR. Interaction network analysis via STRING revealed that the genes , , , and played important roles in the direct interaction with global regulatory genes and the relevant genes of efflux pumps and outer membrane porins. Quantitative reverse transcription-PCR analysis further demonstrated that the increased susceptibility to cephalosporins and quinolones in Δ and Δ mutant cells was accompanied by increased expression of membrane porin genes (, , and ) and reduced expression of efflux pump genes (, , and ), as well as an adverse transcription of the global regulatory genes ( and ). These results indicated that CpxAR and PhoPQ played an important role in the development of MDR in . Enteritidis through regulation of cell membrane permeability and efflux pump activity. . Enteritidis is a predominant Salmonella serotype that causes human salmonellosis and frequently exhibits high-level resistance to commonly used antibiotics, including cephalosporins and quinolones. Although TCSs are known as regulators for bacterial adaptation to stressful conditions, which modulates β-lactam resistance in Vibrio parahaemolyticus and colistin resistance in Salmonella enterica serovar Typhimurium, there is little knowledge of their functional mechanisms underlying the development of antibiotic resistance in . Enteritidis. Here, we systematically identified the TCS elements in . Enteritidis SJTUF12367, revealed that the three TCSs CpxAR, PhoPQ, and GlnGL were crucial for the MDR formation in . Enteritidis, and preliminarily illustrated the regulatory functions of CpxAR and PhoPQ for antimicrobial resistance genes. Our work provides the basis to understand the important TCSs that regulate formation of antibiotic resistance in . Enteritidis.