The type-VII ESX-1 secretion apparatus, encoded by theesx-1genetic locus, is essential for the export of EsxA and EsxB, two major virulence factors ofMycobacterium tuberculosis.ESX-1 also requires the products of the unlinkedespACDoperon for optimal function and these proteins are considered integral parts of the secretion apparatus. Here we show that theespACDoperon is not necessary for the secretion of EspB, another ESX-1 substrate, and this unimpeded secretion of EspB is associated with significant residual virulence. Upon further investigation, we found that purified EspB can facilitateM. tbvirulence even in the absence of EsxA and EsxB, and may do so by binding the bioactive phospholipids phosphatidic acid and phosphatidylserine, both of which are potent bioactive molecules with prominent roles in eukaryotic cell signalling. Our findings provide new insights into the impact of theespACDoperon on the ESX-1 apparatus and reveal a distinct virulence function for EspB with novel implications inM. tb-host interactions.