Competence is an important bioprocess for Streptococcus pneumoniae. Previously, we demonstrated that bacterial second messenger cyclic di-adenosine monophosphate (c-di-AMP) modulates pneumococcal competence. Surprisingly, a low c-di-AMP-producing strain, cdaA*, due to a mutation in the diadenylate cyclase CdaA, is highly susceptible to competence-stimulating peptide (CSP). In this study, we screened cdaA* suppressor mutations resistant to CSP to explore c-di-AMP signaling in S. pneumoniae. A total of 14 clones were sequenced, nine clones possessed mutations in the c-di-AMP phosphodiesterase Pde1, indicating that the susceptibility to CSP of cdaA* is correlated to c-di-AMP levels. Another two clones exhibited a mutation in FabT, a transcription factor controlling cell membrane fatty acid biosynthesis and metabolism. We further showed that deletion of fabT, disruption of the FabT-binding site within the P _fabK promoter, deletion of a fabT activator BriC, or disruption of K ⁺ uptake in cdaA* all rescued the growth defect of cdaA* in media supplemented with CSP. Finally, we found that a c-di-AMP phosphodiesterase-null mutant with high levels of c-di-AMP is highly sensitive to treatment with either ethanol or Triton X-100, which could be corrected by reducing c-di-AMP levels through introducing point mutations in CdaA. Together, these findings indicate that c-di-AMP affects cell membrane integrity.