Two common signal transduction mechanisms used by bacteria to sense and respond to changing environments are two-component systems (TCSs) and eukaryotic-like Ser/Thr kinases and phosphatases (eSTK/Ps). is a Gram-positive bacterium and serious opportunistic pathogen that relies on both a TCS and an eSTK/P pathway for intrinsic resistance to cell wall-targeting antibiotics. The TCS consists of a histidine kinase (CroS) and response regulator (CroR) that become activated upon exposure of cells to cell wall-targeting antibiotics， leading to modulation of gene expression. The eSTK/P pathway consists of a transmembrane kinase (IreK) and its cognate phosphatase (IreP)， which act antagonistically to mediate antibiotic resistance through an unknown mechanism. Because both CroS/R and IreK/P contribute to enterococcal resistance towards cell wall-targeting antibiotics， we hypothesized these signaling systems are intertwined. To test this hypothesis， we analyzed CroR phosphorylation and CroS/R-dependent gene expression to probe the influence of IreK and IreP on CroS/R signaling. In addition， we analyzed the phosphorylation state of CroS which revealed IreK-dependent phosphorylation of a Thr residue important for CroS function. Our results are consistent with a model in which IreK positively influences CroR-dependent gene expression through phosphorylation of CroS to promote antimicrobial resistance in Two-component signaling systems (TCSs) and eukaryotic-like Ser/Thr kinases (eSTKs) are used by bacteria to sense and adapt to changing environments. Understanding how these pathways are regulated to promote bacterial survival is critical for a more complete understanding of bacterial stress responses and physiology. The opportunistic pathogen relies on both a TCS (CroS/R) and an eSTK (IreK) for intrinsic resistance to cell wall-targeting antibiotics. We probed the relationship between CroS/R and IreK， revealing convergence of IreK and the sensor kinase CroS to enhance signaling through CroS/R and increase antimicrobial resistance in This newly described example of eSTK/TCS convergence adds to our understanding of the signaling networks mediating antimicrobial resistance in .