During the infectious cycle, protozoan parasites of the genus Leishmania undergo several adaptive differentiation steps that are induced by environmental factors and crucial for parasite infectivity. Genetic analyses of signaling proteins underlying Leishmania stage differentiation are often rendered difficult due to lethal null mutant phenotypes. Here we used a transgenic strategy to gain insight into the functions of the mitogen-activated Leishmania major protein kinases LmaMPK7 and LmaMPK10 in parasite virulence. We established L. major and Leishmania donovani lines expressing episomal green fluorescent protein (GFP)-LmaMPK7 and GFP-LmaMPK10 fusion proteins. The transgenic lines were normal in promastigote morphology, growth, and the ability to differentiate into metacyclic and amastigote stages. While parasites expressing GFP-LmaMPK10 showed normal infectivity by mouse footpad analysis and macrophage infection assays, GFP-LmaMPK7 transgenic parasites displayed a strong delay in lesion formation and reduced intracellular parasite growth. Significantly, the effects of GFP-LmaMPK7 on virulence and proliferation were due exclusively to protein kinase activity, as the overexpression of two kinase-dead mutants had no effect on parasite infectivity. GFP-LmaMPK7 transgenic L. donovani cells revealed a reversible, stage-specific growth defect in axenic amastigotes that was independent of cell death but linked to nonsynchronous growth arrest and a significant reduction of de novo protein biosynthesis. Our data suggest that LmaMPK7 protein kinase activity may be implicated in parasite growth control and thus relevant for the development of nonproliferating stages during the infectious cycle.