Objective To observe the effect of phosphatidylinositol 3kinase (PI3K) specific inhibitor LY294002 on osteoblast differentiation, and explore the molecular mechanism of PI3K on osteoblast differentiation by regulating PDK-1/Akt signal pathway. Methods Bone marrow mesenchymal stem cells (BMSC) of C57BL/6 mice were cultured in vitro. The maximum safe concentration of LY294002 was detected by CCK-8. BMSC was treated with osteoblast induction medium (OBM) in the control group. BMSC was treated with OBM including LY294002 in the intervention group. The alkaline phosphatase (ALP) activity and mineralization ability of the two groups were detected, and the expression levels of PDK-1/Akt signal pathway and downstream protein and transcription factor genes were detected by Western-Blot and R-PCR, respectively. Results The results of CCK-8 showed that the concentration of 3.2 μM and below had no effect on the proliferation of BMSC, but the effect of LY294002 on osteoblast differentiation in a dose dependent manner. The number of ALP positive cells and extracellular matrix mineralization in the intervention group were significantly lower than those in the control group (P＜0.05). The results of Western-Blot detection showed that the protein expression levels of p-PDK-1 and p-Akt in the intervention group were significantly lower than those in the control group (P＜0.05). The results of RT-PCR showed that the related genes of alkaline phosphatase (ALP) and osteocalcin (OCN) mRNA in the intervention group were significantly lower than those in the control group (P＜0.05). Conclusion Targeted inhibition of PI3K can significantly down-regulate the expression of osteoblastrelated genes ALP and OCN and inhibit osteoblast differentiation through PDK-1/Akt signal pathway.