Synemin is a very unique, large intermediate filament (IF) protein present in all types of muscle cells. It forms heteropolymeric intermediate filaments (IFs) with the major IF proteins desmin and/or vimentin. There are two isoforms of human synemin, [Alpha]-synemin and [Beta]-synemin, which are splice variants from the same gene. Avian synemin has previously been shown to directly interact with the actin-binding and costameric protein [Alpha]-actinin, and with the costameric protein vinculin. Dystrophin, the missing gene product in Duchenne muscular dystrophy (DMD), is enriched at the costameres that are located periodically and subjacent to the sarcolemma of normal skeletal muscle cells. Utrophin is present primarily at the sarcolemma in the neuromuscular and myotendinous junctions (NMJs and MTJs) in normal skeletal muscle cells. Dystrophin and its paralogue, utrophin, belong to the spectrin family of proteins, which include [Beta]-spectrin and [Alpha]-actinin. We show herein that avian synemin directly interacts with both dystrophin and utrophin. Specific regions within both the human synemin isoforms are shown to directly interact with specific domains/regions within the dystrophin and utrophin molecules by protein-protein interaction assays. Blot overlay assays demonstrate that the rod domain of human [Alpha]- and [Beta]-synemin interacts with specific rod domain regions within both dystrophin and utrophin. The C-terminal end of the long tail domain of [Alpha]-synemin interacts with the cysteine-rich (CR) domain of dystrophin, and with the CR domain and one rod domain region of utrophin. The interactions of the rod domain of human synemins with the rod domain regions of both dystrophin and utrophin, and the interaction of the C-terminal end of the tail domain of [Alpha]-synemin with the rod domain region of utrophin have been verified by GST pull-down assays. Mammalian synemin was 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 both the costameric structures located along the sarcolemma and the sarcolemma located within the NMJs and MTJs. These interactions in toto should help maintain the overall stability and integrity of muscle cells.