Eccentric, or lengthening, contractions result in injury and subsequently stimulate the activation and proliferation of satellite stem cells which are important for skeletal muscle regeneration. The discovery of alternative myogenic progenitors in skeletal muscle raises the question as to whether stem cells other than satellite cells accumulate in muscle in response to exercise and contribute to post-exercise repair and/or growth. In this study, stem cell antigen-1 (Sca-1) positive, non-hematopoetic (CD45-) cells were evaluated in wild type (WT) and alpha7 integrin transgenic (alpha7Tg) mouse muscle, which is resistant to injury yet liable to strain, 24 hr following a single bout of eccentric exercise. Sca-1+CD45- stem cells were increased 2-fold in WT muscle post-exercise. The alpha7 integrin regulated the presence of Sca-1+ cells, with expansion occurring in alpha7Tg muscle and minimal cells present in muscle lacking the alpha7 integrin. Sca-1+CD45- cells isolated from alpha7Tg muscle following exercise were characterized as mesenchymal-like stem cells (mMSCs), predominantly pericytes. In vitro multiaxial strain upregulated mMSC stem cells markers in the presence of laminin, but not gelatin, identifying a potential mechanistic basis for the accumulation of these cells in muscle following exercise. Transplantation of DiI-labeled mMSCs into WT muscle increased Pax7+ cells and facilitated formation of eMHC+DiI- fibers. This study provides the first demonstration that mMSCs rapidly appear in skeletal muscle in an alpha7 integrin dependent manner post-exercise, revealing an early event that may be necessary for effective repair and/or growth following exercise. The results from this study also support a role for the alpha7 integrin and/or mMSCs in molecular- and cellular-based therapeutic strategies that can effectively combat disuse muscle atrophy.