The human pathogen Staphylococcus aureus encodes the ESAT6-like Secretion System (ESS). The ESS pathway secretes pathogenic substrates such as EsxA, EsxB, EsxC, EsxD and EssD that mediate staphylococcal establishment in persistent abscess lesions. The biochemical behavior of these substrates is not fully understood. EsxC is species-specific lysine-rich homodimer that lacks recognizable topogenic sequence. Studies have shown that EsxC is required for the secretion of other substrates, thereby revealing its biomedical importance. Here, EsxC self-association was investigated in the presence of several metal ion chelators. Results show that EsxC homodimerization is abolished in the presence of EDTA and EGTA, suggesting a role for calcium in mediating EsxC self-association. Complementation experiments confirm that EsxC homodimerization is calcium-dependent. N- and C-terminal truncations of EsxC were constructed, followed by bacterial two-hybrid screening. Results show that EsxC self-association is mediated by its C-terminal domain. Affinity purification of recombinant EsxC to apparent homogeneity, followed by chemical crosslinking and SDS-PAGE led to the detection of the monomeric and dimeric forms of the protein. In contrast and when a purified EsxC variant lacking the C-terminus was subjected to similar conditions, only the monomeric form was observed. These in vivo and in vitro data highlight the contribution of the C-terminus of the virulence factor EsxC to self-association, and document a previously unreported role for calcium in mediating protein-protein interactions in this pathogenic secretion system.