Xin is a striated muscle-specific actin-binding protein whose mRNA expression has been observed in damaged skeletal muscle. Here we demonstrate increased Xin protein expression early postinjury (≤12 h) and localization primarily to the periphery of damaged myofibers. At 1 day postinjury, Xin is colocalized with MyoD, confirming expression in activated satellite cells (SCs). By 5 days postinjury, Xin is evident in newly regenerated myofibers, with a return to preinjury levels by 14 days of regeneration. To determine whether the increased Xin expression is functionally relevant, tibialis anterior muscles of wild-type mice were infected with Xin-short hairpin RNA (shRNA) adenovirus, whereas the contralateral tibialis anterior received control adenovirus (Control). Four days postinfection, muscles were harvested or injured with cardiotoxin and collected at 3, 5, or 14 days thereafter. When compared with Control, Xin-shRNA infection attenuated muscle regeneration as demonstrated by Myh3 expression and fiber areas. Given the colocalization of Xin and MyoD, we isolated single myofibers from infected muscles to investigate the effect of silencing Xin on SC function. Relative to Control, SC activation, but not proliferation, was significantly impaired in Xin-shRNA-infected muscles. To determine whether Xin affects the G0-G1 transition, cell cycle reentry was assessed on infected C2C12 myoblasts using a methylcellulose assay. No difference in reentry was noted between groups, suggesting that Xin contributes to SC activation by means other than affecting G0-G1 transition. Together these data demonstrate a critical role for Xin in SC activation and reduction in Xin expression results in attenuated skeletal muscle repair.