Staphylococcus aureus contains three members of the LytR-CpsA-Psr (LCP) family of membrane proteins: MsrR, SA0908 and SA2103. The characterization of single-, double- and triple-deletion mutants revealed distinct phenotypes for each of the three proteins. MsrR was involved in cell separation and septum formation and influenced β-lactam resistance; SA0908 protected cells from autolysis; and SA2103, although displaying no apparent phenotype by itself, enhanced the properties of msrR and sa0908 mutants when deleted. The deletion of sa0908 and sa2103 also further attenuated the virulence of msrR mutants in a nematode-killing assay. The severely defective growth phenotype of the triple mutant revealed that LytR-CpsA-Psr proteins are essential for optimal cell division in S. aureus. Growth could be rescued to varying degrees by any one of the three proteins, indicating some functional redundancy within members of this protein family. However, differing phenotypic characteristics of all single and double mutants and complemented triple mutants indicated that each protein played a distinct role(s) and contributed differently to phenotypes influencing cell separation, autolysis, cell surface properties and virulence.