Nucleus pulposus mesenchymal stem cells (NPMSCs) are a potential cell source for intervertebral disc (IVD) regeneration, but little is known about their response to IVD-like high osmolarity (400 mOsm). This study was to investigate the viability, proliferation and protein biosynthesis of nucleus pulposus cells (NPCs), NPMSCs and co-cultured NPMSCs-NPCs under IVD-like high osmolarity conditions. NPCs and NPMSCs were isolated and cultured under standard and IVD-like high osmolarity conditions for 1 or 2 weeks. Cell viability was measured by annexin V-FITC and PI staining, and cell proliferation measured by MTT assay. The expression of SOX-9, aggrecan and collagen-II was measured by RT-PCR and Western blot analyses. IVD-like high osmolarity condition slightly inhibited cell viability and decreased the expression of SOX-9, aggrecan and collagen-II at the mRNA and protein levels in all groups compared with standard condition. NPMSCs could tolerate IVD-like high osmolarity, and NPCs-NPMSCs co-culture increased cell proliferation and the expression of SOX-9, aggrecan and collagen-II under both culture conditions, suggesting that co-culture of NPMSCs-NPCs has potential application for IVD regeneration.