Human umbilical cord-derived mesenchymal stem cells (hUCMSCs) are multipotent cells that have selfrenewal properties and can differentiate into osteocytes, adipocytes, cartilage and extoderm. Bone regeneration and repair are important for the repair of bone injury, skeletal development or continuous remodeling throughout adult life. Thus, investigating the factors influencing osteocyte regeneration from hUCMSCs could be conducive to advancements in skeletal repair and the repair of bone injury. Previous reports have demonstrated that single integrin subunits (alpha V, beta 3, alpha 5) and collagen I contribute to the osteogenic differentiation of human mesenchymal stem cells (hMSCs). However, the functions of the vitronectin receptor alpha V and beta 3 in the osteogenic differentiation of hUCMSCs and bone regeneration remain unclear. Run-related transcription factor 2 (RUNX2) is considered to be an early osteoblastic gene that is upregulated during the osteogenic differentiation of hUCMSCs. Meanwhile, bone sialoprotein (BSP) and collagen I are the most common early markers of osteoblast differentiation. Herein, we found that the mRNA and protein expression of alpha V, beta 3, RUNX2 and collagen I were upregulated during the osteogenic differentiation of hUCMSCs. Overexpression of alpha V and beta 3 in hMSCs increased the levels of RUNX2, BSP, and collagen I, decreased the number of adipocytes and promoted the osteogenic differentiation of hUCMSCs. Meanwhile, down-regulation of alpha V and beta 3 decreased the levels of RUNX2, BSP, and collagen I, increased the number of adipocytes and blocked the osteogenic differentiation of hUCMSCs. In conclusion, the integrin subunits alpha V and beta 3 can promote the osteogenic differentiation of hUCMSCs and encourage bone formation.