C/EBPbeta is a key mediator of cancer-induced skeletal muscle wasting. However, the signaling mechanisms that activate C/EBPbeta in the cancer milieu are poorly defined. Here, we report cancer-induced muscle wasting requires the transcriptional cofactor p300, which is critical for the activation of C/EBPbeta. Conditioned media from diverse types of tumor cells as well as recombinant HSP70 and HSP90 provoked rapid acetylation of C/EBPbeta in myotubes, particularly at its Lys39 residue. Overexpression of C/EBPbeta with mutated Lys39 impaired Lewis lung carcinoma (LLC)-induced activation of the C/EBPbeta-dependent catabolic response, which included upregulation of E3 ligases UBR2 and atrogin1/MAFbx, increased LC3-II, and loss of muscle proteins both in myotubes and mouse muscle. Silencing p300 in myotubes or overexpressing a dominant negative p300 mutant lacking acetyltransferase activity in mouse muscle attenuated LLC tumor-induced muscle catabolism. Administration of pharmacologic p300 inhibitor C646, but not PCAF/GCN5 inhibitor CPTH6, spared LLC tumor-bearing mice from muscle wasting. Furthermore, mice with muscle-specific p300 knockout were resistant to LLC tumor-induced muscle wasting. These data suggest that p300 is a key mediator of LLC tumor-induced muscle wasting whose acetyltransferase activity may be targeted for therapeutic benefit in this disease. SIGNIFICANCE: These findings demonstrate that tumor-induced muscle wasting in mice is abrogated by knockout, mutation of Lys39 or Asp1399, and pharmacologic inhibition of p300.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/7/1331/F1.large.jpg.