Antimicrobial resistance is a widely recognized issue, but less attention has been paid to antimicrobial tolerance. The opportunistic pathogen Enterococcus faecalis, which is responsible for serious hospital-acquired infections, has a high level of intrinsic antibiotic resistance, especially to β-lactam antibiotics. E. faecalis uses the CroRS two-component system to provide antibiotic resistance to cell-wall targeting antibiotics, such as the β-lactam ampicillin. Previous genome profiling studies exploring the CroRS regulon did not show the exact genes responsible for the CroSR dependent ß-lactam resistance. To gain further insights into the mechanistic link between CroRS regulation and β-lactam resistance, we here used experimental evolution to restore ampicillin resistance in a croRS deletion strain. The resulting strains exhibited a 10-fold increase in ampicillin resistance, which is equivalent to that of the wild type. Genome sequencing showed that the only mutations common to all four evolved lines were in genes encoding for enzymes responsible for the synthesis and degradation of the second messenger c-di-AMP. Deletion of croRS led to a marked increase in c-di-AMP levels, while the evolved strains exhibited a decrease in c-di-AMP levels similar to the wild type. Changes in cellular c-di-AMP concentration have been found to be correlated with changes in β-lactam resistance, where excessive second messenger production coincided with β-lactam sensitivity. To gain a better understanding of the mechanistic basis of the correlation between c-di-AMP concentration and β-lactam resistance, targeted mutagenesis of c-di-AMP metabolic genes was performed in combination with phenotypic characterization. These findings will help to improve our understanding of how CroRS regulation controls β-lactam tolerance in E. faecalis.