Antibiotic-resistant enterococci are major causes of hospital-acquired infections. The emergence of Enterococcus faecalis as a significant nosocomial pathogen is a consequence of its inherent resistance to certain antibiotics and of its ability to survive and proliferate in the intestinal tract. Genetic determinants of E. faecalis conferring these properties are largely unknown. Here we show that PrkC, a one-component signaling protein containing a eukaryotic-type Ser/Thr kinase domain, modulates inherent antimicrobial resistance and intestinal persistence of E. faecalis. An E. faecalis mutant lacking PrkC grows at a wild-type rate in the absence of antimicrobial stress but exhibits enhanced sensitivity to cell-envelope-active compounds, including antibiotics that target cell-wall biogenesis and bile detergents. Consistent with its bile sensitivity, the mutant was also impaired at persistence in the intestine of mice. Thus, PrkC regulates key physiological processes in E. faecalis associated with its success as a nosocomial pathogen. The predicted domain architecture of PrkC comprises a cytoplasmic kinase domain separated by a transmembrane segment from extracellular domains thought to bind uncross-linked peptidoglycan, suggesting that PrkC is a transmembrane receptor that monitors the integrity of the E. faecalis cell wall and mediates adaptive responses to maintain cell-wall integrity. Given its role in modulating traits of E. faecalis important for its ability to cause nosocomial infections, we suggest that the one-component signaling protein PrkC represents an attractive target for the development of novel therapies to prevent infections by antibiotic-resistant enterococci.