Skeletal muscle degeneration is a complication arising from a variety of chronic diseases including advanced cancer. Pro-inflammatory cytokine TNF-a plays a pivotal role in mediating cancer-related skeletal muscle degeneration. Here, we show a novel function for retinoblastoma protein (Rb), where Rb causes sarcomeric disorganization. In human skeletal muscle myotubes (HSMMs), up-regulation of cyclin-dependent kinase 4 (CDK4) and concomitant phosphorylation of Rb was induced by TNF-a treatment, resulting in the translocation of phosphorylated Rb to the cytoplasm. Moreover, induced expression of the nuclear exporting signal (NES)-fused form of Rb caused disruption of sarcomeric organization. We identified mammalian diaphanous-related formin 1 (mDia1), a potent actin nucleation factor, as a binding partner of cytoplasmic Rb and found that mDia1 helps maintain the structural integrity of the sarcomere. These results reveal a novel non-nuclear function for Rb and suggest a potential mechanism of TNF-a-induced disruption of sarcomeric organization. DOI: http://dx.doi.org/10.7554/eLife.01228.001