Following skin and the skeleton, skeletal muscle is the most abundant source of the polysaccharide hyaluronan. Despite this, there is little understanding of the role hyaluronan plays in maintenance of skeletal muscle tissue. In distinct mouse and in vitro models of muscle growth and maintenance the three enzymes producing hyaluronan were differentially regulated. During embryonic development, Has2 was the most highly expressed gene transcript relative to housekeeper and increased temporally in association with myogenesis. Although in the mdx mouse model of muscular dystrophy Has3 was the most highly expressed, only Has1 and Has2 expression were significantly different in dystrophic compared to normal muscle. Following myotoxin induced injury, Has3 was the most highly expressed gene and whilst Has1 and Has3 gene expression correlated with the acute inflammatory response, Has2 expression seemed to predominate during periods of myogenic differentiation and the resolution of regeneration. During myogenic differentiation in vitro, Has2 was the most highly expressed and increased as differentiation proceeded. Together this suggested that Has2 may be important for myogenic differentiation, the formation of multinucleated muscle cells. Indeed, siRNA mediated knockdown of Has2 inhibited the fusion of mononucleated C2C12 myoblasts into multinucleated myotubes and altered proteoglycan transcript expression. Loss of cell associated hyaluronan and pericellular matrix associated hyaluronan was observed with both Has2 siRNA and the hyaluronan synthesis inhibitor 4-methylumbelliferone and could explain the inhibition of fusion by preventing required cell-cell interactions or intracellular signaling mediated by the pericellular matrix. Inhibition of fusion by Has2 siRNA could not be rescued by addition of soluble high molecular weight hyaluronan, further indicating the necessity for intrinsic hyaluronan synthesis by myoblasts. This has important consequences for muscle pathologies where aberrant inflammation and ineffective regeneration contribute to a devastating loss of function, implicating roles for Has1 and Has3 in inflammation and Has2 in muscle cell regeneration.