Bone morphogenetic protein and activin membrane-bound inhibitor (BAMBI) is a transforming growth factor β (TGF-β) type I receptor antagonist that negatively regulates TGF-β and bone morphogenetic protein (BMP) signaling. BAMBI has been shown to be regulated by TGF-β signaling; however, whether BAMBI can be regulated by BMP signaling remains to be determined. The aim of this study was to investigate the effect of BMP2 on the regulation of BAMBI expression in human granulosa-lutein cells and the underlying mechanisms. Both primary and immortalized human granulosa-lutein cells were used as research models. Using dual inhibition approaches, our results showed that BMP2 activated SMAD1/5/8 phosphorylation and up-regulated BAMBI mRNA levels, which was reversed by the BMP type I receptor inhibitors, DMH-1 and dorsomorphin, but not by SB431542 (activin/TGF-β type I receptor inhibitor). Moreover, the combined knockdown of SMAD1 and SMAD5 completely abolished the BMP2-induced up-regulation of BAMBI. Similarly, knockdown of SMAD4 reversed the BMP2-induced up-regulation of BAMBI. Pre-treatment with BMP2 inhibited the TGF-β1-induced phosphorylation of SMAD2/3 and up-regulation of MMP2, and these inhibitory effects were reversed by knockdown of endogenous BAMBI. Our findings indicate that BAMBI is a BMP-responsive gene and that BAMBI participates in the negative feedback regulation of TGF-β signaling in the human ovary.