We tested the hypothesis that cytokines derived from differentiated skeletal muscle cells in culture induce neutrophil chemotaxis after mechanical strain. Flexible-bottom plates with cultured human muscle cells attached were exposed to mechanical strain regimens (ST) of 0, 10, 30, 50, or 70 kPa of negative pressure. Conditioned media were tested for the ability to induce chemotaxis of human blood neutrophils in vitro and for a marker of muscle cell injury (lactate dehydrogenase). Conditioned media promoted neutrophil chemotaxis in a manner that was related both to the degree of strain and to the magnitude of muscle cell injury (ST 70 > ST 50 > ST 30). Protein profiling using a multiplex cytokine assay revealed that mechanical strain increased the presence of IL-8, granulocyte-macrophage colony-stimulating factor (GM-CSF), granulocyte colony-stimulating factor, monocyte chemotactic protein (MCP)-1, and IL-6 in conditioned media. We also detected 14 other cytokines in conditioned media from control cultures that did not respond to mechanical strain. Neutralization of IL-8 and GM-CSF completely inhibited the chemotactic response for ST 30 and ST 50 and reduced the chemotactic response for ST 70 by 40% and 47%, respectively. Neutralization of MCP-1 or IL-6 did not reduce chemotaxis after ST 70. This study enhances our understanding of the immunobiology of skeletal muscle by revealing that skeletal muscle cell-derived IL-8 and GM-CSF promote neutrophil chemotaxis after injurious mechanical strain.