Muscle wasting during sepsis and other catabolic conditions is, at least in part, mediated by glucocorticoids and is associated with upregulated transcription of multiple genes in the ubiquitin‐proteasome proteolytic pathway. In addition to transcription factors, nuclear cofactors, including p300, regulate gene transcription. We tested the hypothesis that glucocorticoids upregulate the expression of p300 in muscle cells. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with dexamethasone resulted in a dose‐ and time‐dependent increase in p300 protein and mRNA levels. Surprisingly, the effect of dexamethasone on p300 levels was not inhibited by the glucocorticoid receptor (GR) antagonist RU38486 and RU38486 exerted an agonist effect on p300, increasing its expression. Co‐immunoprecipitation showed that treatment of the myotubes with dexamethasone resulted in protein–protein interaction between p300 and C/EBPβ, but not C/EBPδ. The present results suggest that glucocorticoids upregulate the expression of p300 and its interaction with C/EBPβ in skeletal muscle. Increased expression and activity of p300 may be involved in the regulation of gene transcription in glucocorticoid‐dependent muscle wasting. J. Cell. Biochem. 94: 1058–1067, 2005. © 2005 Wiley‐Liss, Inc.