To explore the role of Brucella BI-1 in Brucella suis S2, we constructed the Brucella BI-1 deletion mutant strain and its complementary strain. We then determined the effect of Brucella BI-1 deletion on the physiological characteristics of Brucella suis S2 and revealed them via integrated transcriptomic and proteomic analyses. Brucella BI-1 deletion altered the membrane properties of Brucella suis S2 and decreased its resistance to acidic pH, H2O2, polymyxin B, and lincomycin. Additionally, deleting Brucella BI-1 led to defective growth, cell division, and viability in Brucella suis S2. In conclusion, our results revealed that Brucella BI-1 is a bacterial cytoprotective protein involved in membrane homeostasis, cell division, and stress resistance in Brucella suis S2.