Aim To investigate Glial cell line-derived neurotrophic factor (GDNF) expression in normal and wounded rat periodontal ligament (PDL) and the effects of GDNF on human PDL cells (HPDLCs) migration and extracellular matrix expression in HPDLCs. Material and Methods The expression of GDNF and GDNF receptors was examined by immunocyto/histochemical analyses. Gene expression in HPDLCs treated with GDNF, interleukin-1 beta (IL-1 beta), or tumour necrosis factor-alpha (TNF-a) was quantified by quantitative RT-PCR (qRT-PCR). In addition, we examined the migratory effect of GDNF on HPDLCs. Results GDNF was expressed in normal rat PDL and cultured HPDLCs. HPDLCs also expressed GDNF receptors. In wounded rat PDL, GDNF expression was up-regulated. QRT-PCR analysis revealed that IL-1 beta and TNF-a significantly increased the expression of GDNF in HPDLCs. Furthermore, GDNF induced migration of HPDLCs, which was blocked by pre-treatment with the peptide including Arg-Gly-Asp (RGD) sequence, or neutralizing antibodies against integrin aV beta 3 or GDNF. Also, GDNF up-regulated expression of bone sialoprotein (BSP) and fibronectin in HPDLCs. Conclusions GDNF expression is increased in rat wounded PDL tissue and HPDLCs treated with pro-inflammatory cytokines. GDNF enhances the expression of BSP and fibronectin, and migration in an RGD-dependent manner via the integrin aV beta 3. These findings suggest that GDNF may contribute to wound healing in PDL tissue.