Nucleus pulposus-derived mesenchymal stem cells (NPMSCs) have shown a good prospect in the regeneration of intervertebral disc (IVD) tissues. However, fresh NPMSCs are not always readily available for basic research and clinical applications. Therefore, there is a need for an effective long-term cryopreservation method for NPMSCs. The aim of this study was to determine whether adding icariin (ICA) to the conventional cryoprotectant containing dimethyl sulfoxide (DMSO) had a better cryoprotective effect for NPMSCs. The results showed that the freezing solution containing ICA along with DMSO significantly increased the postthawed cell viability, decreased the apoptosis rate, improved cell adherence, and maintained the mitochondrial functions, as compared to the freezing solution containing DMSO alone. And the inhibition of oxidative stress and upregulation of heat shock proteins (HSPs) in the presence of ICA also confirmed the beneficial effect of ICA. Furthermore, ICA had no cytotoxicity and did not alter the characteristics of postthawed NPMSCs. In conclusion, these results suggested that the addition of ICA to the conventional freezing medium could improve the viability and function of the cryopreserved human NPMSCs and provided an optimal formulated freezing solution for human NPMSC cryopreservation.