The B-RAF kinase plays an important role both in tumor induction and maintenance in several cancers. The molecular basis of the inactive B-RAF(WT) and B-RAF(V600E) inhibition and selectivity of a series of inhibitors was examined with a combination of molecular dynamics (MD), free energy MM-PBSA and local-binding energy (LBE) approaches. The conformational stability of the unbounded kinases and in particular the processes of the B-RAF (V600E) mutant activation were analyzed. A unique salt bridge network formed mainly by the catalytic residues was identified in the unbounded B-RAFs. The reorganization of this network and the restriction of the active segment flexibility upon ligand binding inhibit both B-RAF(WT) and B-RAF (V600E), thus appearing as an important factor for ligand selectivity. A significant correlation between the binding energies of the compounds in B-RAF(WT) and their inhibition effects on B-RAF (V600E) was revealed, which can explain the low mutant selectivity observed for numerous inhibitors. Our results suggest that the interactions between the activation segment and the alpha C-helix, as well as between the residues in the salt bridge network, are the major mechanism of the B-RAF (V600E) activation. Overall data revealed the important role of Lys601 for ligand activity, selectivity and protein stabilization, proposing an explanation of the observed strong kinase activation in the K601E mutated form.