Escherichia coli K1 invasion of human brain microvascular endothelial cells (HBMEC) mediated by outer membrane protein A (OmpA) results in the leakage of HBMEC monolayers. Despite the influence of nitric oxide (NO) in endothelial cell tight junction integrity, its role in E. coli-induced HBMEC monolayer permeability is poorly defined. Here, we demonstrate that E. coli invasion of HBMEC stimulates NO production by increasing the inducible nitric oxide synthase (iNOS) expression. Exposure to NO-producing agents enhanced the invasion of OmpA(+)E. coli and thereby increased the permeability of HBMEC. OmpA(+)E. coli-induced NO production lead to increased generation of cGMP and triggered the expression of OmpA receptor, Ec-gp96 in HBMEC. Pre-treatment of HBMEC with iNOS inhibitors or by introducing siRNA to iNOS, but not to eNOS or cGMP inhibitors abrogated the E. coli-induced expression of Ec-gp96. Overexpression of the C-terminal truncated Ec-gp96 in HBMEC prevented NO production and its downstream effector, cGMP generation and consequently, the invasion of OmpA(+)E. coli. NO/cGMP production also activates PKC-alpha, which is previously shown to be involved in HBMEC monolayer leakage. These results indicate that NO/cGMP signalling pathway plays a novel role in OmpA(+)E. coli invasion of HBMEC by enhancing the surface expression of Ec-gp96.