Haematogenous spread is a key step in the development of Acanthamoeba granulomatous encephalitis, however it is not clear how circulating amoebae cross the blood-brain barrier to enter the CNS to produce disease. Using the primary human brain microvascular endothelial cells (HBMEC), which constitute the blood-brain barrier, here it is shown that Acanthamoeba abolishes the HBMEC transendothelial electrical resistance. Using traversal assays, it was observed that Acanthamoeba crosses the HBMEC monolayers. The primary interactions of Acanthamoeba with the HBMEC resulted in increased protein tyrosine phosphorylations and the activation of RhoA, suggesting host-parasite cross-talk. Furthermore, Western blot assays revealed that Acanthamoeba degraded occludin and zonula occludens-1 proteins in a Rho kinase-dependent manner. Overall, these findings suggest that Acanthamoeba affects the integrity of the monolayer and traverses the HBMEC by targeting the tight junction proteins.