17β-estradiol (E2) mediates a wide variety of complex biological processes. While E2 effects on the reproductive system are quite well established, less is known about how it affects the physiology of other tissues. Skeletal muscle expresses both estrogen receptor isoforms (ERα and ERβ). In addition, female rat muscles show fewer histopathological changes after repeated eccentric contractions than male muscles; ovariectomized female rats exhibit higher indexes of exercise-induced muscle membrane damage which disappear in after estradiol treatment. the mechanisms underlying the role played by E2 and remain still elusive. In actively proliferating rat myoblast cells (L6) (grown in 10% serum), E2 rapidly increased the glucose transporter type 4 (Glut-4) translocation at the cell membranes; successively, the increase of well known differentiation markers of myogenesis (i.e., myogenin and myosin heavy chain, MHC) was evidenced by Western blot analysis at 6 and 24 h, respectively, after E2 stimulation. Seven days after E2 treatment the alignment and fusion of L6 myoblasts into multinucleated myotubes were visible. Next we compared the effect of E2 and IGF-I. Both hormones influenced the three muscle differentiation markers considered, even if the E2 efficacy was less than IGF-I. In addition, E2 effects were completely prevented by the pure ER inhibitor. The contribute of both nuclear and extra-nuclear action mechanisms on E2-induced modification of differentiation markers was thus evaluated by pre-trating cells with cycloheximide, actinomycin, and the palmitoylation inhibitor 2-Br, that prevents the membrane starting signals of both receptor isoforms. These data indicate that both E2-dependent rapid signals and nuclear action are required for in E2-induced L6 differentiation. In particular, ERα-dependent AKT activation is necessary to control the first step of myogenic differentiation. Moreover, both receptors mediate the E2-induced activation of p38 which, in turn, affects the expression of myogenin and MHC. The contribute of ERα in activating the previously identified E2-dependent effects has been evaluated by using both ERα and ERβ selective agonists (PPT and DPN, respectively) and by reducing ERα with specific siRNAs. As expected, E2 was unable to induce AKT phosphorylation, whereas the E2-dependent activation of p38 was still present, in ERα-silenced cells. Intriguingly, E2 was not able any more to induce the Glut-4 translocation and the increase of myogenin and MHC level in ERα-depleted cells, thus confirming the pivotal role of ERα in the L6 differentiation. All together these data indicate that E2, like other extra-cellular growth factors, modulates specific cell signals affecting the skeletal muscle development providing the basis of gender-related physiological differences in skeletal muscle recovery after damage.