Abstract C/EBPβ is a key mediator of cancer-induced skeletal muscle wasting. However, the signaling mechanisms that activate C/EBPβ 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/EBPβ. Conditioned media from diverse types of tumor cells as well as recombinant HSP70 and HSP90 provoked rapid acetylation of C/EBPβ in myotubes, particularly at its Lys39 residue. Overexpression of C/EBPβ with mutated Lys39 impaired Lewis lung carcinoma (LLC)–induced activation of the C/EBPβ-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.