Abstract: OBJECTIVE To observe the impact of two-component system (TCS) regulator gene ompR on virulence of Acinetobacter baumannii and observe its regulatory mechanisms. METHODS The ompR gene knockout strain AB43ΔompR was constructed in A. baumannii AB43 strain by homologous recombination. Then ompR gene was ligated into an expression vector and complemented into the knockout strain to generate the complementary strain. All strains were verified by PCR amplification. Growth curves were determined for the wild-type, knockout, and complementary strains. The Galleria mellonella infection model was employed to evaluate changes in virulence, and cell invasion assays were conducted to examine the adhesion and invasion capabilities of A. baumannii against A549 epithelial cells. The changes in membrane structure were assessed by three types of fluorescent probes: Propidium iodide(PI), 1-N-phenyl-naphtylamine(NPN) and 3,3'-Dipropylthiadicarbocyanine iodide(DiSC3(5)). The adenosine triphosphate(ATP)level was measured by luciferase bioluminescence method. RESULTS The ompR gene knockout and complementary strains were successfully constructed, and the growth rate of the knockout strain was lower than that of the wild-type strain. The result of G. mellonella infection experiment showed that the survival rate of larvaes infected with the knockout strain was remarkably higher than that of those infected with the wild-type strain. The result of in vitro cell experiment indicated that the invasion capability of the knockout strain against the epithelial cell decreased remarkably(P＜0.05). The result of examination of membrane structure showed that the permeability of the outer membrane increased, and the inner membrane was damaged(P＜0.05). The result of ATP quantification assay revealed that the intracellular ATP level of the knockout strain remarkably declined(P＜0.05). CONCLUSIONS The knockout TCS regulator ompR gene can reduce the virulence of A. baumannii, and it reduces the survival of bacteria in the host environment by on one hand decreasing the bacterial invasion ability and on other hand destructing the cell membrane integrity and the energy metabolism homeostasis. The study not only provides novel theoretical bases for understanding the mechanisms of A. baumannii infection, but also suggests that the ompR gene may serve as a potential target for developing novel antibacterial drugs, offering new ideas for clinical treatment of multidrug-resistant organisms infections.