Neisseria meningitidis, an obligate human pathogen, remains a leading cause of meningitis and fatal sepsis. Meningococci are known to secrete a family of proteins, such as FrpC, with sequence similarity to the repeat-in-toxin (RTX) proteins via the type I secretion system. The meningococcal type I secretion proteins are encoded at two distant genetic loci, NMB1400 (hlyB) and NMB1738/1737 (hlyD/tolC), and are separated from the RTX toxin-like substrates. We have characterized the promoter elements of both hlyB and hlyD by primer extension and lacZ reporter fusions and revealed the growth phase-dependent upregulation of both genes. In addition, we showed that the MisR/MisS two-component system negatively regulates the expression of hlyB and hlyD/tolC. Direct binding of MisR to hlyB and hlyD promoters was demonstrated by electrophoretic mobility shift assay (EMSA), and DNase I protection assays identified MisR binding sites overlapping the promoter elements. Direct repression of hlyB transcription by MisR was supported by in vitro transcription assays. Mutations in the MisR/S system affected, but did not eliminate, the growth phase-dependent upregulation of hlyB, suggesting additional regulatory mechanisms. Increased secretion of RTX toxin-like proteins was detected in the cell-free media from misS mutant cultures, indicating that the amounts of extracellular RTX toxin-like proteins are, in part, controlled by the abundance of the type I secretion apparatus. This is, to our knowledge, the first example of a two-component system mediating secretion of cytotoxin family proteins by controlling expression of the type I secretion proteins.