During early pregnancy, trophoblast cells are exposed to relatively low-oxygen tension. Recently, the Rho GTPase family has been shown to play a key role in hypoxia-inducible factor-1 (HIF-1) α induction in renal cell carcinoma. The present study was designed to investigate the effect of low-oxygen conditions on RhoA expression in trophoblast cells isolated from early stages of human placenta and in trophoblast-derived BeWo cells and JAR cells. Immunoblot and RT-PCR analyses showed that low-oxygen conditions (1% O₂ or 250 μm CoCl₂) stimulated expression of RhoA protein and mRNA. Pull-down assays demonstrated that these low-oxygen conditions increased RhoA activity. Preincubation of BeWo cells with Clostridium botulinum C3 exoenzyme, a specific inhibitor of Rho, inhibited hypoxia-induced HIF-1α expression. Under 1% O₂ or 250 μm CoCl₂, BeWo cells, transfected with a dominant-negative RhoA, exhibited decreased levels of HIF-1α protein and mRNA compared with the control vector transfectants. BeWo cells expressing constitutively active RhoA showed enhanced protein levels of not only HIF-1α but also vascular endothelial growth factor (VEGF) and glucose transporter 1, which are target gene products of HIF-1α. These findings suggest that up-regulation of RhoA induced by low-oxygen conditions may play an important role in regulation of HIF-1α expression in trophoblast cells.