Affixin/β-parvin is an integrin-linked kinase (ILK)-binding focal adhesion protein highly expressed in skeletal muscle and heart. To elucidate the possible role of affixin in skeletal muscle, we established stable C2C12 cell line expressing T7-tagged human affixin (C2C12-affixin cells). Exogenous expression of affixin promotes lamellipodium formation where affixin, ILK αp21-activated kinase (PAK)-interactive exchange factor (PIX) and βPIX accumulate. The association of affixin and βPIX was confirmed by immunoprecipitation and pull down assay. In C2C12-affixin cells, an increased level of activated Rac1 but not Cdc42 was observed, and mutant βPIX lacking guanine nucleotide exchange factor activity inhibited lamellipodium formation. These results suggest that affixin is involved in reorganization of subsarcolemmal cytoskeletal actin by activation of Rac1 through α and βPIXs in skeletal muscle. STRUCTURED SUMMARY: