Objective: To explore effect of the isoquercetin on osteoblast differentiation and expression level of HDAC1 and Runx2. Methods: The type 1 collagen (Col1), alkaline phosphatase (ALP) and osteopontin (OPN) expression level in MC3T3-E1 cells was detected by using qRT-PCR after treated with 1×10⁻⁸, 1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵ mol/L isoquercetin. The ALP activity was assayed using ALP detection kit. Mineralization of MC3T3-E1 was evaluated by Alizarin red staining. What’s more, the mRNA levels of HDAC1 and Runx2 in MC3T3-E1 cells were observed by using qRT-PCR post-treatment by 1×10⁻⁸, 1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵ mol/L isoquercetin. Results: The Col 1, ALP and OPN expression level turned significantly higher after treated with 1×10−⁸, 1×10⁻⁷, 1×10⁻⁶, 1×10⁻⁵ mol/L isoquercetin (P<0.05). There was a certain dose effect between them. As the same, the ALP activity and mineralization of MC3T3-E1 was higher post-exposure to the isoquercetin. HDAC1 in the MC3T3-E1 cells had lower expression level after treated with the isoquercetin (P<0.05). However, Runx2 in the MC3T3-E1 cells had higher expression level after exposed to the isoquercetin (P<0.05). Conclusion: The isoquercetin can promote the expression of Runx2 through inhibiting the expression of HDAC1, in order to improve the differentiation of osteoblast. The results provide a new theoretical basis for the application of isoquercetin in the treatment of osteoporosis.