The periodontal ligament (PDL) is specialized connective tissue fibers for attaching a tooth to the alveolar bone and for supporting the tooth to withstand the mechanical stress occurred during chewing and continuous orthodontic tooth movement. These mechanical stresses induce physiological processes such as inflammatory response, periodontal tissue and bone remodeling. However, the mechanism of hypo-osmotic stress-induced cellular response in PDL cells remains poorly understood. We hypothesized that receptor activator of nuclear factor-kappaB (NF-κB) ligand (RANKL) and osteoprotegerin (OPG) released from PDL cells under hypo-osmotic stress regulate bone remodeling, and investigated the mechanisms of RANKL and OPG expression by primary cultured human PDL cells in response to hypo-osmotic stress. Hypo-osmotic stress increased the mRNA expression of RANKL but not OPG as well as intracellular calcium concentration ([Ca2+]i). This increases in [Ca2+]i was completely inhibited by gadolinium and lanthanum, non-specific plasma membrane Ca2+ channel blockers. We found TRPM3 and TRPV4 expressed in primary cultured human PDL cells and activities of these channels of TRPM3 and TRPV4 were confirmed by a whole-cell patch-clamp technique. Finally, aminoethoxydiphenyl borate and ruthenium red, each blockers of TRPM3 and TRPV4, reduced hypo-osmotic stress-induced increases in [Ca2+]i and RANKL mRNA expression. These results suggest that hypo-osmotic stress induces increases in [Ca2+]i through TRPM3 and TRPV4 to regulate RANKL mRNA expression in primary cultured human PDL cells.