The ESX-1 (ESAT-6 system 1) secretion system plays a conserved role in the virulence of diverse mycobacterial pathogens, including the human pathogen and , an environmental mycobacterial species. The ESX-1 system promotes the secretion of protein virulence factors to the extracytoplasmic environment. The secretion of these proteins triggers the host response by lysing the phagosome during macrophage infection. Using proteomic analyses of the secretome in the presence and absence of a functional ESX-1 system, we and others have hypothesized that MMAR_2894, a PE family protein, is a potential ESX-1 substrate in We used genetic and quantitative proteomic approaches to determine if MMAR_2894 is secreted by the ESX-1 system, and we defined the requirement of for ESX-1-mediated secretion and virulence. We show that MMAR_2894 is secreted by the ESX-1 system in and is itself required for the optimal secretion of the known ESX-1 substrates in Moreover, we found that MMAR_2894 was differentially required for hemolysis and cytolysis of macrophages, two lytic activities ascribed to the ESX-1 system. Both , the cause of human tuberculosis (TB), and , a pathogen of ectotherms, use the ESX-1 secretion system to cause disease. There are many established similarities between the ESX-1 systems in and in Yet the two bacteria infect different hosts, hinting at species-specific functions of the ESX-1 system. Our findings demonstrate that MMAR_2894 is a PE protein secreted by the ESX-1 system of We show that MMAR_2894 is required for the optimal secretion of mycobacterial proteins required for disease. Because the gene is not conserved in , our findings demonstrate that MMAR_2894 may contribute to a species-specific function of the ESX-1 system in , providing new insight into how the and systems differ.