Appropriate trophoblast invasion into the maternal endometrium is essential for successful human implantation and placentation. Connective tissue growth factor (CTGF), also known as CCN2, is a matricellular protein that is expressed in the placenta. Interestingly, the CTGF expression levels in the placenta and serum from patients with severe preeclampsia or fetal growth restriction are higher than those from healthy controls. However, to date, the role of CTGF in the regulation of trophoblast cell invasion remains unclear. Transforming growth factor-β1 (TGF-β1) is a potent stimulator of CTGF expression and has been shown to inhibit trophoblast cell invasiveness. However, whether CTGF mediates TGF-β1-inhibited human trophoblast cell invasion is unknown. In the present study, we show that treatment with TGF-β1 upregulates CTGF expression in a human trophoblast cell line, HTR-8/SVneo, and in primary human trophoblast cells. Our results also demonstrate that the SMAD2/3 signaling pathways are required for TGF-β1-induced upregulation of CTGF. Importantly, CTGF knockdown attenuates TGF-β1-inhibited cell invasion. Furthermore, cell invasiveness is decreased by treatment with recombinant CTGF. These results provide evidence that CTGF mediates TGF-β1-inhibited human trophoblast cell invasion.