Francisella tularensis contains four putative acid phosphatases that are conserved in Francisella novicida. An F. novicida quadruple mutant (AcpA, AcpB, AcpC, and Hap [DeltaABCH]) is unable to escape the phagosome or survive in macrophages and is attenuated in the mouse model. We explored whether reduced survival of the DeltaABCH mutant within phagocytes is related to the oxidative response by human neutrophils and macrophages. F. novicida and F. tularensis subspecies failed to stimulate reactive oxygen species production in the phagocytes, whereas the F. novicida DeltaABCH strain stimulated a significant level of reactive oxygen species. The DeltaABCH mutant, but not the wild-type strain, strongly colocalized with p47(phox) and replicated in phagocytes only in the presence of an NADPH oxidase inhibitor or within macrophages isolated from p47(phox) knockout mice. Finally, purified AcpA strongly dephosphorylated p47(phox) and p40(phox), but not p67(phox), in vitro. Thus, Francisella acid phosphatases play a major role in intramacrophage survival and virulence by regulating the generation of the oxidative burst in human phagocytes.