Nicotine, the main psychoactive component of tobacco, affects cell metabolism, proliferation, adhesion and, importantly, the osteogenic differentiation of fibroblasts. Approximately 15% of all orthodontic patients are adults among who one-fifth are smokers. Hence, it is necessary to have insight into the effects of nicotine on the osteogenic differentiation of hPDLCs during orthodontic tooth movement. This study aimed to investigate the effects and mechanisms of nicotine on the osteogenic differentiation of human periodontal ligament cells (hPDLCs) under the application of cyclic tensile stress. hPDLCs were obtained from donor third molars. The hPDLCs were treated with nicotine and/or cyclic tensile stress that was applied with a cell stress plus unit. The effect of nicotine on cell viability was analyzed using the MTT assay. The osteogenic differentiation of hPDLCs was detected by alkaline phosphatase staining, Alizarin Red S staining, quantitative real-time polymerase chain reaction and western blotting. In combination with cyclic tensile stress, nicotine prevented the tensile stress-induced increase in alkaline phosphatase activity, formation of mineralization nodules and the upregulation of mRNA and protein expression of Runt-related transcription factor 2, transcription factor Sp7 and collagen type I; however, canonical Wnt pathway was activated. Furthermore, the addition of Dickkopf-related protein 1 and α-bungarotoxin counteracted the negative effect of nicotine and rescued the osteogenic differentiation of hPDLCs, respectively. These results indicate that nicotine prevents the increased osteogenic potential of hPDLCs induced by cyclic tensile stress by binding to an α7 nicotinic acetylcholine receptor and activating the canonical Wnt pathway.