To assess the influence of high extracellular glucose levels on the osteogenic differentiation of bone marrow stromal cells (BMSCs) and to determine if Sonic hedgehog (Shh) protein can alleviate those effects. BMSCs were incubated with NG (normal glucose), NG+Shh (200 ng/ml Shh in normal glucose), NG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in normal glucose), HG (high glucose), HG+Shh (200 ng/ml Shh in high glucose), and HG+Shh+Gan (200 ng/ml Shh and 5 micromol/L GANT61 in high glucose). The expression levels of Shh signaling pathway genes Patched 1 (PTCH1) and osteogenesis-related genes were tested, which included bone morphogenetic protein 4 (BMP4), runt-related transcription factor 2 (Runx2), and osteopontin (OPN). Alkaline phosphatase (ALPase) activity and mineralized matrix formation were also investigated. Immunofluorescent staining of Gli1 was tested for Shh signaling activation. We found that recombinant Shh in normal-glucose medium could promote osteogenic differentiation of BMSCs, while inhibiting Shh signaling by GANT61 could antagonize this differentiation. Besides that high glucose impaired the Shh signaling as well as osteogenic differentiation of BMSCs, reactivation of Shh signal pathway by addition of Shh protein could mitigate the inhibition while further deactivation by Shh inhibitor GANT61 could retain their osteogenic inhibitions. The above data suggest that Shh pathway activity is involved in the HG condition mediated osteoblastic differentiation deficiency for BMSCs and that recombinant Shh could alleviate this inhibitory effect.