AbstractLeishmania spp. are obligate intracellular parasites that inhabit the phagolysosomes of macrophages. Manipulation of host cell signaling pathways and gene expression by Leishmania is critical for Leishmania’s survival and resultant pathology. Here, we show that infection of macrophages with Leishmania promastigotes in vitro causes specific cleavage of the NF‐κB p65RelA subunit. Cleavage occurs in the cytoplasm and is dependent on the Leishmania protease gp63. The resulting fragment, p35RelA, migrates to the nucleus, where it binds DNA as a heterodimer with NF‐κB p50. Importantly, induction of chemokine gene expression (MIP‐2/CXCL2, MCP‐1/CCL2, MIP‐1α/CCL3, MIP‐1β/CCL4) by Leishmania is NF‐κB dependent, which implies that p35RelA/p50 dimers are able to activate transcription, despite the absence of a recognized transcriptional transactivation domain. NF‐κB cleavage was observed following infection with a range of pathogenic species, including L. donovani, L. major, L. mexicana, and L. (Viannia) braziliensis, but not the non‐pathogenic L. tarentolae or treatment with IFN‐γ. These results indicate a novel mechanism by which a pathogen can subvert a macrophage's regulatory pathways to alter NF‐κB activity.