The aim of the present study is to examine whether low level Ga-Al-As laser irradiation has an effect on hedgehog signaling pathway during osteoblast proliferation in vitro. The molecular mechanism of how low level Ga-Al-As laser irradiation promotes bone formation is explored. This study provides a theoretical basis for the clinical application of low level Ga-Al-As laser irradiation in oral implant. Mouse osteoblastic cell line MC3T3-E1 is cultured in vitro. The cultures after laser irradiation are treated with recombinant N-terminals sonic hedgehog (N-Shh) or hedgehog inhibitor cyclopamine respectively. The experiment is divided into 4 groups. The proliferation activity is detected by cell counting, MTS, flow cytometry at 12, 24, 48, 72 h after laser irradiation. Proliferation activity of laser irradiation and N-Shh group is remarkably increased compared with those of laser irradiation group. Proliferation activity of laser irradiation and cyclopamine group is remarkably decreased compared with those of laser irradiation group, meanwhile proliferation activity of laser irradiation and cyclopamine group is remarkably increased compared with those of control group. These results suggest that low level Ga-Al-As laser irradiation activate hedgehog signaling pathway during osteoblast proliferation in vitro. hedgehog signaling pathway is one of the signaling pathways by which low level Ga-Al-As laser irradiation regulates osteoblast proliferation.