Chondrocytes located in hyaline cartilage may maintain phenotype while the chondrocytes situated in calcified cartilage differentiate into hypertrophy. Chondrogenic and hypertrophic differentiation of mesenchymal stem cells (MSCs) are two subsequent processes during endochondral ossification. However, it is necessary for chondrocytes to hold homeostasis and to inhibit hypertrophic differentiation in stem cell-based regenerated cartilage. Dihydroartemisinin (DHA) is derived from artemisia apiacea which has many biological functions such as anti-malarial and anti-tumor. Whereas the effects of DHA on chondrogenic and hypertrophic differentiation are poorly understand. In this study, the cytotoxicity of DHA was determined by CCK8 assay and the cell apoptosis was analyzed by flow cytometry. Additionally, the effects of DHA on chondrogenic and hypertrophic differentiation of MSCs are explored by RT-PCR, western blotting and immunohistochemistry. The results showed that DHA inhibited expression of chondrogenic markers including Sox9 and Col2a1 by activating Nrf2 and Notch signaling. After induced to chondrogenesis, cells were treated with hypertrophic induced medium with DHA. The results revealed that hypertrophic markers including Runx2 and Col10a1 were down-regulated following DHA treatment through Pax6/HOXA2 and Gli transcription factors. These findings indicate that DHA is negative to chondrogenesis and is protective against chondrocyte hypertrophy to improve chondrocytes stability. Therefore, DHA might be not suited for chondogenesis but be potential as a new therapeutic candidate to maintain the biological function of regenerated cartilage.