We have shown that Acanthamoeba spp. activate TLR4 on corneal epithelial cells and induce secretion of chemokines. However, the components of Acanthamoeba trophozoites that induce chemokines production remain unknown. We sought to identify the trophozoite molecules that interact with TLR4 on human corneal epithelial (HCE) cells and trigger IL-8 production. Acanthamoeba membrane protein (AcMP) was isolated by homogenization of trophozoites. The supernatants were collected, solubilized, and membrane fractions were separated by centrifugation using Mem-PERTM plus kit. To examine functional activity of AcMP, HCE and TLR4-expressing HEK293 cells were incubated with or without A. castellanii (1×105 cells/ml) and AcMP (10, 25, and 50 µg/ml) for 24 hours. AcMP was chromatographed by fast protein liquid chromatography (FPLC) and fractions were pooled into four peaks (AcMP-P1 - AcMP-P4). TLR4-ligand in AcMP-P1 - AcMP-P4 was determined by Western blotting. HEK293 and HCE cells were incubated with or without A. castellanii, lipopolysaccharide (LPS, 10 µg/ml), and AcMP-P1 - AcMP-P4 (20 µg/ml) for 24 hours. qRT-PCR and ELISA were used to examine the ability of AcMP-P1 - AcMP-P4 to stimulate IL-8 production in HEK293 and HCE cells. Inhibition of TLR4 involved preincubating HEK293 and HCE cells for 1 hour with neutralizing TLR4-antibody (10 µg/ml) or with the control antibody (10 µg/ml, goat serum) followed by incubation with or without A. castellanii, LPS, and AcMP-P2 for 24 hours. AcMP induced significant IL-8 production at doses of 10, 25, and 50 µg/ml in HEK293 cells while IL-8 mRNA expression and IL-8 secretion were significantly increased in HCE cells at the dose of 50 µg/ml. Treatments of HEK293 with FPLC chromatographed trophozoites’ proteins, AcMP-P1 - AcMP-P4; only AcMP-P2 upregulated significant IL-8 production and mRNA expression. Western blotting of AcMP-P1 - AcMP-P4 showed TLR4-antigen in AcMP-P2 and was recognized an approximate 15-kDa protein band. Anti-TLR4 antibody attenuated IL-8 secretion that is stimulated by AcMP-P2 from HEK293 and HCE cells. These results suggest that A. castellanii trophozoites recognize TLR4 on HCE and HEK293 cells by an approximate 15-kDa molecular mass protein of AcMP and induce IL-8 secretion.