Skeletal muscles express estrogen receptor (ER) α and ERβ. However, the roles of estrogens acting through the ERs in skeletal muscles remain unclear. The effects of 17β-estradiol (E2) on myogenesis were studied in C2C12 myoblasts. E2 and an ERα-selective agonist propylpyrazole-triol depressed myosin heavy chain (MHC), tropomyosin, and myogenin levels and repressed the fusion of myoblasts into myotubes. ER antagonist ICI 182,780 cancelled E2-repressed myogenesis. E2 induced ubiquitin-specific peptidase 19 (USP19) expression during myogenesis. E2 replacement increased USP19 expression in the gastrocnemius and soleus muscles of ovariectomized mice. Knockdown of USP19 inhibited E2-repressed myogenesis. Mutant forms of USP19 lacking deubiquitinating activity increased MHC and tropomyosin levels. E2 decreased ubiquitinated proteins during myogenesis, and the E2-decreased ubiquitinated proteins were increased by knockdown of USP19. Propylpyrazole-triol increased USP19 expression, and ICI 182,780 inhibited E2-increased USP19 expression. Overexpression of ERα or knockdown of ERβ enhanced the effects of E2 on the levels of USP19, MHC, and tropomyosin, whereas knockdown of ERα, overexpression of ERβ, or an ERβ-selective agonist diarylpropionitrile abolished their effects. A mutant form of ERα that is constitutively localized in the nucleus increased USP19 expression and decreased MHC and tropomyosin expression in the presence of E2. Furthermore, in skeletal muscle satellite cells, E2 inhibited myogenesis and increased USP19 expression, and diarylpropionitrile repressed E2-increased USP19 expression. These results demonstrate that (i) E2 induces USP19 expression through nuclear ERα, (ii) increased USP19-mediated deubiquitinating activity represses myogenesis, and (iii) ERβ inhibits ERα-activated USP19 expression.