The objective of the present study was to investigate the effect of thymosin β4 (Tβ4) on the wound healing of rat palatal (RP) mucosa and related cellular properties. Cell viability, adhesion and migration of primary cultured RP cells were observed in the presence of Tβ4 at various concentrations ranging from 1 to 1,000 ng/ml. The mRNA and protein expression of matrix metalloproteinase 2 (MMP2) and vascular endothelial growth factor (VEGF) in Tβ4-treated RP cells was assessed by quantitative polymerase chain reaction (RT-qPCR) and western blot analysis, respectively. For the in vivo assay, Tβ4 was applied to excisional wounds (3 mm in diameter) that were made in the center of the palate (n=6). Images of the wound areas were captured and assessed histologically one week after surgery. Tβ4 did not affect cell viability and adhesion, but RP cell migration was stimulated by Tβ4 at concentrations of 100 and 1,000 ng/ml. Tβ4 also increased the mRNA and protein expression of MMP2 and VEGF in RP cells. In the animal model, palatal wound closure was significantly enhanced in rats treated with Tβ4. The results of the present study indicated that Tβ4 promotes the wound healing of RP mucosa. Enhancement of RP cell migration and angiogenesis is likely to be involved in the promotion of wound healing.