Objective To explore the mechanism of recombinant thymosin β4 (Tβ4) accelerating skin wound heal ing in rats by regulating laminin 5 expression. Methods Two full thickness 8 mm punch wounds were made at the costovertebralangle on dorsal surface of each adult male rats weighing 200-250 g. Sixty rats were randomized into the control group (n=15) and the experimental group (n=45), which was subdivided into low, medium and high dose groups (n=15). Tβ4 was appl ied topically at 2, 6, 18 μg in 50 μL PBS for every 12 hours after model making in the experimental group. The identical amounts of phosphate buffered sal ine was appl ied in the control group. Wound heal ing was observed after model making and immunohistochemical observation was conducted 2, 4 and 7 days after operation. Results Seven days after operation, wound contracted obviously and most of the wounds connected well with the margin. In the control group, low dose group, medium dose group and high dose group, the wound heal ing rate were 7.67% ± 5.46%, 29.01% ± 7.43%, 26.54% ± 11.49% and 10.39% ± 3.96% respectively 2 days after operation; 28.16% ± 13.76%, 37.99% ± 13.05%, 42.00% ± 9.56% and 39.58% ± 12.74% respectively 4 days after operation; 59.08% ± 19.02%, 64.15% ± 17.92%, 77.39% ± 8.45% and 69.78% ± 8.45% respectively 7 days after operation. At 2 days after operation, significant differences were notified in heal ing rats between 3 sub-experimental groups and the control group (P lt; 0.05). Immunohistochemistry staining showed that there was a l ittle more positive expression of laminin 5 2 days after operation that beneficial to promote the prol iferation and differentiation of cell in every group, including positive cells andECM. But in medium group there was fewer expression, only at the borderl ine and bottom of the wound, while the expression significantly increased 4 days after operation (P lt; 0.05) and there was a relative high expression 7 days after operation (P lt; 0.01). Conclusion Tβ4 can inhibit the expression of laminin 5 early, and then up-regulate laminin 5 expression to moderate the reformation of ECM, promote the migration of epidemic cell and accelerate skin wound heal ing.