Objective：To investigate the role of RhoA-ROCK signal pathway in the reorganization of actin cytoskeleton and the changes of morphology induced by hypoxia in breast cancer cells（MDA-MB-435 cells）. Methods：During the experiments the breast cancer cells were exposed to hypoxia for 16 h or pre-incubated with a special inhibitor of ROCK, Y-27632（10 μmol／L） for 1h before hypoxia in vitro. The reorganization of cellular actin cytoskeleton and the changes of cellular morphology induced by hypoxia or pretreatment of Y-27632 were examined by confocal laser scanning microscopy. Results：Hypoxia induced the depolymerization of actin cytoskeleton, ɑｎｄ the changes of arrangement and distribution of stress fibers, and the changes of morphology were observed obviously in breast cancer cells. Pretreatment with Y-27632（10 μmol／L） before hypoxia could prevent the depolymerization of actin cytoskeleton and the changes of arrangement and distribution of stress fibers induced by hypoxia in breast cancer cells. Conclusion：RhoA-ROCK signal pathway may mediate the reorganization of actin cytoskeleton and morphological changes in breast cancer cells during hypoxia.