Insulin stimulates osteoblast proliferation and differentiation as an anabolic agent in bone. Insulin Receptor Tyrosine Kinase Substrate (IRTKS) is involved in insulin signaling as an adapter for insulin receptors (IR). Here, we showed that IRTKS levels were significantly decreased in bone marrow mesenchymal stem cells (BMSCs) derived from the bone marrow of patients with osteoporosis. Based on relevant experiments, we observed that IRTKS promoted the proliferation, migration, and osteoblast differentiation of BMSCs and MC3T3-E1 cells. In addition, we identified a Phosphatase and Tensin homolog deleted on chromosome 10 (PTEN) as a potential active substrate of IRTKS. We demonstrated a direct interaction between IRTKS and PTEN using co-immunoprecipitation. Subsequently, we confirmed that the SH3 domain of IRTKS directly binds to the C-terminal tail of PTEN. Further experimental results demonstrated that PTEN attenuated the promoting effects of IRTKS on the proliferation, migration, and osteoblast differentiation of BMSCs and MC3T3-E1 cells. In conclusion, this study suggests that IRTKS contributes to osteogenic differentiation by inhibiting PTEN phosphorylation and provides a potential therapeutic target for osteoporosis patients.