To investigate the protein expression of myogenin, a mark of muscle regeneration, and TGF-beta1, an important factor of myelofibrosis, in long-term denervated posterior cricoarytenoid muscles (PCAMs) in order to find the appropriate time point of reinnervation in long-term denervated PCAMs.Thirty-eight patients with vocal paralysis were divided into four groups, the 0.5-year denervation group, the 1-year denervation group, the 2-year denervation group and the over 3-year denervation group. Twelve normal adults served as a control group. The change in expression of related factors such as myogenin and TGF-beta1 were observed using immunofluorescence stain and Western blot.Immunostaining with antibody against myogenin showed no staining in innervated, however, by 0.5-year of muscle denervation, there was a significant accumulation of myogenin protein in myonuclei. There was a peak in expression of myogenin in 1-year denervated muscles. After 2 years of denervation, expression of myogenin protein in myonuclei was decreased, but after 3 years of denervation, no expression of myogenin protein in myonuclei was found. TGF-beta1 showed no staining in innervated, however, by 0.5-year and 1-year of muscle denervation there was a significant accumulation of TGF-beta1 protein in endochylema of myofiber, After 2 years of denervation, expression of TGF-beta1 protein in endochylema of myofiber was decreased, but after 3 years of denervation, no expression of TGF-beta1 protein in endochylema could be found. In Western blot, the change in protein expression of myogenin was observed 7.12-fold (F = 332.205, P < 0.001) increase from 0.5-year denervated muscles to innervated muscles, 15.58-fold (P < 0.001) increase from 1-year denervated muscles to innervated muscles, 6.50-fold (P < 0.001) increase from 2-years denervated muscles to innervated muscles (P < 0.001). After 3 years, the protein expression of myogenin decreased, 1.12-fold to innervated muscles. The change in protein expression of TGF-beta1 was observed 4.80-fold (F = 106.192, P < 0.001) increase from 0.5-year denervated muscles to innervated muscles, 10.59-fold (P < 0.001) increase from 1-year denervated muscles to innervated muscles, 6.01-fold (P < 0.001) increase from 2-years denervated muscles to innervated muscles(P < 0.001). After 3 years, the protein expression of TGF-beta1 decreased 1.20-fold to innervated muscles. There was significant positive correlation between expression of myogenin and TGF-beta1 in long-term denervated PCAMs.The change in expression of myogenin and TGF-beta1 indicated that there was a good muscles regeneration and a high amplitude of myelofibrosis within 2 years.