The physiological hypoxic condition favors the angiogenesis in the placenta. However, it remains unclear how hypoxia regulates the invasion of human extravillous trophoblast cells. In the present study, we first showed that alpha5 integrin expression increased and alpha1 integrin expression decreased in human extravillous trophoblast cells cultured in 1% oxygen as compared with control cells cultured in 8% oxygen. Further data showed that the neutralizing antibody against alpha5 integrin increased the invasion of human extravillous trophoblast cells and the neutralizing antibody against alpha1 integrin inhibited the invasion of human extravillous trophoblast cells. Human extravillous trophoblast cells cultured in 1% oxygen showed reduced invasive capacity, which can be effectively blocked by alpha5 integrin neutralizing antibody. Moreover, human extravillous trophoblast cells exposed to 1% oxygen demonstrated increased expression of transforming growth factor-beta3 (TGFB3), and recombinant human TGFB3 inhibited the invasion of human extravillous trophoblast cells in a dose-dependent manner. The neutralizing antibodies against alpha5 integrin and TGFB3 markedly abrogated hypoxia-induced invasion inhibition in human extravillous trophoblast cells. These data indicate that hypoxia may inhibit the invasion of human extravillous trophoblast cells through inducing the integrin switch from alpha1 integrin to alpha5 integrin and promoting TGFB3 expression.