ABSTRACTStreptococcus suisis an recognized zoonotic pathogen of swine and severely threatens human health. Zinc is the second most abundant transition metal in biological systems. Here, we investigated the contribution of zinc to the drug resistance and pathogenesis ofS. suis. We knocked out the genes of AdcACB and Lmb, two Zn-binding lipoproteins. Compared to the wild-type strain, we found that the survival rate of this double-mutant strain (ΔadcAΔlmb) was reduced in Zinc-limited medium, but not in Zinc-supplemented medium. Additionally, phenotypic experiments showed that the ΔadcAΔlmbstrain displayed impaired adhesion to and invasion of cells, biofilm formation, and tolerance of cell envelope-targeting antibiotics. In a murine infection model, deletion of theadcAandlmbgenes inS. suisresulted in a significant decrease in strain virulence, including survival rate, tissue bacterial load, inflammatory cytokine levels, and histopathological damage. These findings show that AdcA and Lmb are important for biofilm formation, drug resistance, and virulence inS. suis.IMPORTANCETransition metals are important micronutrients for bacterial growth. Zn is necessary for the catalytic activity and structural integrity of various metalloproteins involved in bacterial pathogenic processes. However, how these invaders adapt to host-imposed metal starvation and overcome nutritional immunity remains unknown. Thus, pathogenic bacteria must acquire Zn during infection in order to successfully survive and multiply. The host uses nutritional immunity to limit the uptake of Zn by the invading bacteria. The bacterium uses a set of high-affinity Zn uptake systems to overcome this host metal restriction. Here, we identified two Zn uptake transporters inS. suis, AdcA and Lmb, by bioinformatics analysis and found that anadcAandlmbdouble-mutant strain could not grow in Zn-deficient medium and was more sensitive to cell envelope-targeting antibiotics. It is worth noting that the Zn uptake system is essential for biofilm formation, drug resistance, and virulence inS. suis. The Zn uptake system is expected to be a target for the development of novel antimicrobial therapies.