Ewing sarcoma is a rare but aggressive disease most common in young adults. This cancer is driven by a unique chimeric fusion oncogene but targeted strategies have been elusive. Here we report the identification of the protein kinase PKC-ß (PRKCB) as a disease-specific druggable target for treatment of Ewing sarcoma. We found that transcriptional activation of PRKCB was directly regulated by the chimeric fusion oncogene EWSR1-FLI1 that drives this cancer. PRKCB phosphorylated histone H3T6 to permit global maintenance of H3K4 trimethylation at a variety of gene promoters. PRKCB loss induced apoptosis in vitro and prevented tumor growth in vivo. Gene expression profiling revealed a strong overlap between genes modulated by EWSR1-FLI1 and PRKCB in regulating crucial signaling pathways. Taken together, our findings offer a preclinical proof-of-concept for PRKCB as a promising therapeutic target in Ewing sarcoma. Cancer Res; 72(17); 4494–503. ©2012 AACR.