AbstractNucleus 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.