Abstract Acanthamoeba spp. are opportunistic pathogens that cause granulomatous amebic encephalitis. We compared the highly pathogenic species A. culbertsoni to the relatively less pathogenic species A. castellanii for its capacity to elicit from neonatal rat microglia the gene expression of pro-inflammatory cytokines. Acanthamoeba culbertsoni elicited a robust cytokine gene response by neonatal rat microglia in vitro as compared to A. castellanii. The preponderant cytokine elicited at the mRNA and protein levels was interleukin-1β. In addition, transmission electron microscopy revealed that microglial cells were capable of phagocytozing A. castellanii. In contrast, A. culbertsoni destroyed microglia. Collectively, these results suggest that a combined action of pro-inflammatory cytokines and destruction of host cells by amebae contribute to the pathology caused by the more pathogenic species.