Wnt5a, a member of the noncanonical Wnt proteins, is known to play important roles in the development of various organs and in postnatal cell functions. However, little is known about the effects of Wnt5a on human periodontal ligament (PDL) cells. In this study, we examined the localization and potential function of Wnt5a in PDL tissue. Immunohistochemical analysis revealed that Wnt5a was expressed predominantly in rat PDL tissue. Semi‐quantitative reverse‐transcription polymerase chain reaction and Western blotting analysis demonstrated that human PDL cells (HPDLCs) expressed Wnt5a and its receptors (Ror2, Fzd2, Fzd4, and Fzd5). Removal of occlusal pressure by extraction of opposing teeth decreased Wnt5a expression in rat PDL tissue, and the expression of Wnt5a and its receptors in HPDLCs was upregulated by exposure to mechanical stress. Stimulation with Wnt5a significantly enhanced the proliferation and migration of HPDLCs. Furthermore, Wnt5a suppressed osteoblastic differentiation of HPDLCs cultivated in osteogenic induction medium, while it significantly enhanced the expression of PDL‐related genes, such as periostin, type‐I collagen, and fibrillin‐1 genes, and the production of collagen in HPDLCs cultivated in normal medium. Both knockdown of periostin gene expression by siRNA and inhibition of TGFβ1 function by neutralizing antibody suppressed the Wnt5a‐induced PDL‐related gene expression and collagen production in HPDLCs. Interestingly, in HPDLCs cultured with Wnt5a, TGFβ1 neutralizing antibody significantly suppressed periostin expression, while periostin siRNA had no effect on TGFβ1 expression. These results suggest that Wnt5a expressed in PDL tissue plays specific roles in inducing collagen production by PDL cells through TGFβ1‐mediated upregulation of periostin expression. J. Cell. Physiol. 9999: 2647–2660, 2015. © 2015 Wiley Periodicals, Inc.