Raf kinases are important cancer drug targets. Paradoxically, many B-Raf inhibitors induce the activation of Raf kinases. Cryo-electron microscopy structural analysis of a phosphorylated B-Raf kinase domain dimer in complex with dimeric 14-3-3, at a resolution of similar to 3.9 angstroms, shows an asymmetric arrangement in which one kinase is in a canonical "active" conformation. The distal segment of the C-terminal tail of this kinase interacts with, and blocks, the active site of the cognate kinase in this asymmetric arrangement. Deletion of the C-terminal segment reduces Raf activity. The unexpected asymmetric quaternary architecture illustrates how the paradoxical activation of Raf by kinase inhibitors reflects an innate mechanism, with 14-3-3 facilitating inhibition of one kinase while maintaining activity of the other. Conformational modulation of these contacts may provide new opportunities for Raf inhibitor development.