Acanthamoeba castellanii is a free-living ubiquitous amoeba, with a worldwide distribution, that can occasionally infect humans, causing particularly severe infections in immune compromised individuals. Dissecting the immunology of Acanthamoeba infections has been considered problematic due to the very low incidence of disease despite the high exposure rates. Whilst macrophages are acknowledged as playing a significant role in Acanthamoeba infections little is known about how this facultative parasite influences macrophage activity. Therefore, in this study we investigate the effects of Acanthamoeba on the activation of resting macrophages. Consequently, murine bone marrow derived macrophages were co-cultured with trophozoites of either the laboratory Neff strain, or a clinical isolate of A. castellaniiIn vitro real-time imaging demonstrated that trophozoites of both strains often established evanescent contact with macrophages. Both Acanthamoeba strains induced a pro-inflammatory macrophage phenotype characterized by significant production of IL-12 and IL-6. However, macrophages co-cultured with the clinical isolate of Acanthamoeba produced significantly less IL-12 and IL-6 in comparison to the Neff strain. The utilization of macrophages derived from MyD88, TRIF, TLR2, TLR4, TLR2/4 deficient mice indicated that Acanthamoeba-induced pro-inflammatory cytokine production was through MyD88-dependent, TRIF independent, TLR4-induced events. This study shows for the first time the involvement of TLRs, expressed on macrophages in the recognition and response to Acanthamoeba trophozoites.