MprAB and PhoPR are important two-component systems (TCSs) in Mycobacterium tuberculosis, and both regulate EspR, a key regulator of the ESX-1 secretion system. Although previous studies suggest that the response regulator PhoP does not directly regulate mprA, the interplay between MprAB and PhoPR remains unclear. In this study, we found that the response regulator MprA can bind to the phoP promoter. Four repeat motifs, D1-D4, constituting two predicted binding sites, were located in the region protected by MprA in DNA footprinting. D1-D4 lack the reported conserved MprA binding sequences, indicating that MprA can recognize a greater range of target sites. Interestingly, D1-D2 overlap a previously reported PhoP binding site, and mutation of D1-D2 inhibited PhoP binding, whereas the D3-D4 site, but not the D1-D2 site, was required for MprA binding. EMSA assays also suggest that MprA and PhoP compete to bind to the phoP promoter. The results of the transcriptional and western blot assays are consistent with a model in which MprA positively controls the phoP expression, which in turn upregulates the expression of espR. These findings reveal complex regulation of a major mycobacterial TCS by dual TCSs.