Introduction: Bronchopulmonary dysplasia (BPD) is a chronic lung disorder that is most common among children born prematurely, is characterized with airway hyperreactivity and lung injury. Rho/Rho-kinase signaling recently has received an increased attention in airway diseases. ----- Aims: The aim of the study was to determine the role of Rho/Rho-kinase in pathophysiology of experimental BPD and the effect of pharmacologically targeting of this pathway. ----- Materials and methods: At the 4th day of life rat pups were exposed either to hyperoxia (>95% O2) or room air for 7 days. Some sets of animals during exposure received i.p. Rho-kinase inhibitors Y-27632 or fasudil. At 12th day of life animals were euthanized and tracheal cylinders were prepared for in vitro force measurement to study contraction of tracheal smooth muscle (TSM) toward different doses of methacholine (MCh); epithelial growth factor or prostaglandin F2α in absence or presence of Y-27632 (10 μM) or fasudil (10 μM). For relaxant responses preparations were pre-contracted with bethanechol then treated with electrical field stimulation (EFS) in absence or presence of Y-27632 or fasudil. ----- Results: In hyperoxia-exposed animals the contractile responses of TSM toward MCh were significantly increased (P<0.01) compared to room air-exposed animals, and the maximal values of contractile responses were 1.8 ± 0.17 g in hyperoxia group and 1.13 ± 0.15 g, respectively. Treatment with Rho-kinase inhibitors either in vitro or in vivo reversed this effect of hyperoxia and normalized responses to control level. Relaxant responses of TSM were significantly decreased (P<0.001) in hyperoxic group of animals compared with room air group, and maximal values of relaxant responses (at 20V) were 38.80 ± 4.20% and 82.02 ± 6.20 % at 20V, respectively. Treatment with Rho-kinase inhibitors either in vitro or in vivo restored the impaired relaxation in hyperoxic group to normal level. Hyperoxia potentiated the contractile effect of exogenous EGF and PGF2α on TSM, while presence of Rho-kinase inhibitors reversed the contractile responses induced by these agonists. ----- Conclusion: This study prove that Rho/Rho-kinase signaling plays an important role in airway hyperreactivity induced by hyperoxia, and pharmacological targeting of this pathway provides an effective therapeutic approach to prevent the adverse effects of neonatal hyperoxia.