Leishmania pathogenesis is primarily studied using the disease‐inducing promastigote stage of Leishmania major. Despite many efforts, all attempts so far have failed to culture the disease‐relevant multiplying amastigote stage of L. major. Here, we established a stably growing axenic L. major amastigote culture system that was characterized genetically, morphologically, and by stage‐specific DsRed protein expression. We found parasite stage‐specific disease development in resistant C57BL/6 mice. Human neutrophils, as first host cells for promastigotes, do not take up amastigotes. In human macrophages, we observed an amastigote‐specific complement receptor 3‐mediated, endocytotic entry mechanism, whereas promastigotes are taken up by complement receptor 1‐mediated phagocytosis. Promastigote infection of macrophages induced the inflammatory mediators TNF, CCL3, and CCL4, whereas amastigote infection was silent and resulted in significantly increased parasite numbers: from 7.1 ± 1.4 (after 3 h) to 20.1 ± 7.9 parasites/cell (after 96 h). Our study identifies Leishmania stage‐specific disease development, host cell preference, entry mechanism, and immune evasion. Since the amastigote stage is the disease‐propagating form found in the infected mammalian host, the newly developed L. major axenic cultures will serve as an important tool in better understanding the amastigotedriven immune response in leishmaniasis.—Wenzel, U. A., Bank, E., Florian, C., Förster, S., Zimara, N., Steinacker, J., Klinger, M., Reiling, N., Ritter, U., van Zandbergen, G. Leishmania major parasite stage‐dependent host cell invasion and immune evasion. FASEB J. 26, 29–39 (2012). www.fasebj.org