This study aimed to investigate the synergy between transforming growth factor beta 3 (TGF-β3) and insulin-like growth factor 1 (IGF-1) on nucleus pulposus-derived mesenchymal stem cells (NP-MSCs) and the underlying mechanism using a serum-free culture system. NP-MSC proliferation and viability were measured using a CCK-8 assay and annexin V-FITC/propidium iodide, respectively. NP-MSCs in micromasses were investigated for differentiation towards nucleus pulposus cells (NPCs). SOX-9, collagen-I, collagen-II, aggrecan and decorin expressions were detected by RT-PCR and immunoblotting. Matrix deposition was assessed by sulfated glycosaminoglycan (sGAG) analysis. Novel chondrogenic and nucleus pulposus (NP) genes were detected to distinguish differentiated cell types. MAPK/ERK and TGF/Smad signaling pathways were also examined. As a result, the synergy between TGF-β3 and IGF-1 enhanced NP-MSC viability, extracellular matrix (ECM) biosynthesis and differentiation towards NPCs, partly through the activation of the MAPK/ERK signaling pathway. Therefore, the synergy between TGF-β3 and IGF-1 ameliorates NP-MSC viability, differentiation and promotes intervertebral disc regeneration.