By coprecipitation, we identified RNA-binding proteins in the Gram-positive opportunistic pathogen Enterococcus faecalis known to be deficient of the RNA chaperone Hfq. We particularly characterized one belonging to the Cold-shock Protein (Csp) family (Ef2925) renamed CspR for Cold-shock protein RNA-binding. Compared to the wild-type strain, the ΔcspR mutant was less virulent in an insect infection model (Galleria mellonella), exhibited decreased persistence in mouse kidneys and lower survival inside peritoneal macrophages. As expected, we found that the ΔcspR mutant strain was more impaired in its growth than the parental strain in cold condition and in its long-term survival under nutrients starvation. All these phenotypes were restored after complementation of the ΔcspR mutant. In addition, Western blot analysis showed that CspR was over-expressed under cold-shock condition as well as in stationary phase. Since CspR may act as an RNA chaperone, putative targets were identified using a global proteomic approach completed with transcriptomic assays. This study revealed that 19 proteins were differentially expressed in the ΔcspR strain (9 upregulated, 10 downregulated) and that CspR mainly acted at the post-transcriptional level. These data highlight for the first time the role of the RNA-binding protein CspR as a regulator in E. faecalis and its requirement in stress response and virulence in this important human pathogen.