Human mesenchymal stem cells (MSCs) have the potential to differentiate into nucleus pulposus (NP)-like cells under specific stimulatory conditions. Thus far, the effects of bone morphogenetic protein 3 (BMP3) and the cocktail effects of BMP3 and transforming growth factor (TGF)- on MSC proliferation and differentiation remain obscure. Therefore, this study was designed to clarify these unknowns. MSCs were cultured with various gradients of BMP3 and BMP3/TGF-, and compared with cultures in basal and TGF- media. Cell proliferation, glycosaminoglycan (GAG) content, gene expression, and signaling proteins were measured to assess the effects of BMP3 and BMP3/TGF- on MSCs. Cell number and GAG content increased upon the addition of BMP3 in a dose-dependent manner. The expression of COL2A1, ACAN, SOX9, and KRT19 increased following induction with BMP3 and TGF-, in contrast to that of COL1A1, ALP, OPN, and COMP. Smad3 phosphorylation was upregulated by BMP3 and TGF-, but BMP3 did not affect the phosphorylation of extracellular-signal regulated kinase (ERK) 1/2 or c-Jun N-terminal kinase (JNK). Our results reveal that BMP3 enhances MSC proliferation and differentiation into NP-like cells, as indicated by increased cell numbers and specific gene expressions, and may also cooperate with TGF- induced positive effects. These actions are likely related to the activation of TGF- signaling pathway.