Tropomodulin (Tmod) is a cytoskeletal actin-capping protein that interacts with tropomyosin at the pointed end of actin filaments. E-Tmod is an isoform that expresses predominantly in cardiac cells and slow skeletal muscle fibers. We unexpectedly discovered significant levels of Tmod in nuclei and then defined peptide domains in Tmod responsible for nuclear import and export. These domains resemble, and function as, a nuclear export signal (NES) and a pattern 4 nuclear localization signal (NLS). Both motifs are conserved in other Tmod isoforms and across species. Comparisons of wild-type Tmod and Tmod carrying mutations in these peptide domains revealed that Tmod normally traffics through the nucleus. These observations logically presuppose that Tmod functions may include a nuclear role. Indeed, increasing Tmod in the nucleus severely hampered myogenic differentiation and selectively suppressed muscle-specific gene expression (endogenous p21, myosin heavy chain, myogenin, and Tmod) but did not affect endogenous glyceraldehyde-3-phosphate dehydrogenase or expression from a transfected E-GFP vector. These results suggest that, at least in myogenic cells, nuclear Tmod may be involved in the differentiation process.