Regulation of cell renewal in the periodontium is a critical cell function that has not been clarified. Sonic hedgehog (Shh) is a secreted signaling molecule that plays a key role during development and adult tissue homeostasis. In the present study, we have analyzed the role played by Shh in human periodontal ligament stem cell (HPLSC) proliferation. HPLSC were isolated with anti-STRO-1 antibodies. Shh increased the expression of GLI1 and PTC-1 and selectively stimulated cell proliferation in STRO-1(+) derived from adult periodontal ligament. Shh components were localized to primary cilia in STRO-1(+) cells after Shh stimulation. STRO-1(+) also expressed Shh, suggesting an autocrine-regulated phenomenon. Thus, we propose that Shh plays a critical role in the regulation of cell proliferation in STRO-1(+)/HPLSC.