Recent data suggest a physiological role for the proinflammatory cytokine TNF-α in skeletal muscle regeneration. However, the underlying mechanism is not understood. In the present study, we analyzed TNF-α-activated signaling pathways involved in myogenesis in soleus muscle injured by cardiotoxin (CTX) in TNF-α receptor double-knockout mice (p55^−/−p75^−/−). We found that activation of p38MAPK, which is critical for myogenesis, was blocked in CTX-injured p55^−/−p75^−/− soleus on day 3 postinjury when myogenic differentiation was being initiated, while activation of ERK1/2 and JNK MAPK, as well as transcription factor NF-κB, was not reduced. Consequently, the phosphorylation of transcription factor myocyte enhancer factor-2C, which is catalyzed by p38 and crucial for the expression of muscle-specific genes, was blunted. Meanwhile, expression of p38-dependent differentiation marker myogenin and p21 were suppressed. In addition, expression of cyclin D1 was fivefold that in wild-type (WT) soleus. These results suggest that myogenic differentiation is blocked or delayed in the absence of TNF-α signaling. Histological studies revealed abnormalities in regenerating p55^−/−p75^−/− soleus. On day 5 postinjury, new myofiber formation was clearly observed in WT soleus but not in p55^−/−p75^−/− soleus. To the contrary, p55^−/−p75^−/− soleus displayed renewed inflammation and dystrophic calcification. On day 12 postinjury, the muscle architecture of WT soleus was largely restored. Yet, in p55^−/−p75^−/− soleus, multifocal areas of inflammation, myofiber death, and myofibers with smaller cross-sectional area were observed. Functional studies demonstrated an attenuated recovery of contractile force in injured p55^−/−p75^−/− soleus. These data suggest that TNF-α signaling plays a critical regulatory role in muscle regeneration.