The 800-nm diode laser is used clinically for hair removal and leg vein clearance. However, the effects of the laser on skin collagen synthesis have not been established. This study aims to research whether the 800-nm laser can be used for non-ablative rejuvenation and its possible mechanism by using an animal model. Eight 2-month-old rats were irradiated with the 800-nm diode laser at 20, 40, and 60 J/cm(2), respectively. Skin samples were taken for histological study and dermal thickness measurement at day 30 after laser irradiation. The expression of procollagen type I, III, IV, transforming growth factor-β (TGF-β), Smad2, 3, 4, and phosphorylated-Smad2, 3 in the rat skin was analyzed 24 h after completing all laser treatments by using RT-PCR and Western blot. Immunohistochemistry was performed to evaluate the content of type I collagen in the skin at day 30 after laser irradiation. The 800-nm diode laser treatments markedly improved the histological structure and increased dermal thickness compared to the non-irradiated controls. Laser irradiation at 40 J/cm(2) significantly up-regulated the expression of procollagen type I and IV, TGF-β and Smad2, 3, 4. The p-Smad2 and p-Smad3 levels were also enhanced in the laser-irradiated skin. The 800-nm laser is effective in improving skin structure and inducing skin new collagen expression. New collagen synthesis induced by the 800-nm laser was mediated by TGF-β/Smad signaling pathway. Thus, it seemed that the 800-nm laser could be used for non-ablative rejuvenation in the future.