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Nucleus pulposus cell (NPC) transplantation can be a potential therapeutic approach for intervertebral disc degeneration (IDD). However, low cell viability has restricted the therapeutic capacity of NPCs, and sources of natural NPCs are limited. Bone marrow-derived mesenchymal stem cells (BMSCs) and adipose-derived mesenchymal stem cells (ADSCs) can be differentiated toward NPC-like cells. However, it is unknown whether there are differences in the abilities of these two cell types to differentiate into NPC-like cells, or which cell type exhibits the best differentiation ability. The present study compared the abilities of BMSCs and ADSCs to differentiate toward NPC-like cells with or without a 3D culture system to lay a foundation for stem cell transplantation therapy for IDD. BMSCs were isolated from the rat whole bone marrow cell using the repeated adherent culture method. ADSCs were isolated from rat adipose tissues in the subcutaneous inguinal region using the enzyme digestion method. Cells were identified using flow cytometry. Cell viability was assessed via Cell Counting Kit-8 assays, and reverse transcription-quantitative PCR and western blotting were carried out to evaluate the expression of NPC markers and chondrocyte-specific genes. Glycosaminoglycans (GAGs) and proteoglycans were examined via Alcian blue and safranin O staining, respectively. ADSCs in 3D culture displayed the highest cell proliferative ability, compared with the 2D culture system and BMSC culture. In addition, ADSCs in 3D culture exhibited increased GAG and proteoglycan synthesis than BMSCs. Compared with BMSCs in 3D culture, ADSCs in 3D culture exhibited higher mRNA and protein expression of NPC marker genes (hypoxia-inducible factor 1-α, glucose transporter 1) and chondrocyte-specific genes (Sox-9, aggrecan and type II collagen). The present findings indicated that ADSCs exhibited a better ability to differentiate into NPC-like cells in 3D culture compared with BMSCs, which may be of value for the regeneration of intervertebral discs using cell transplantation therapy. [ABSTRACT FROM AUTHOR]