Synemin is a unique, very large intermediate filament (IF) protein present in all types of muscle cells, which forms heteropolymeric intermediate filaments (IFs) with the major IF proteins desmin and/or vimentin. We show herein that tissue-purified avian synemin directly interacts with both dystrophin and utrophin, and that specific expressed regions of both of the mammalian (human) synemin isoforms (α-synemin and β-synemin) directly interact with specific expressed domains/regions of the dystrophin and utrophin molecules. Mammalian synemin is also shown to colocalize with dystrophin within muscle cell cultures. These results indicate that synemin is an important IF protein in muscle cells that helps fortify the linkage between the peripheral layer of cellular myofibrils and the costameric regions located along the sarcolemma and the sarcolemma region located within the neuromuscular and myotendinous junctions (NMJs and MTJs).