Advanced computational techniques including transition path sampling and free energy calculations are combined synergistically to reveal the activation mechanism at unprecedented resolution for a small signaling protein, chemotaxis protein Y. In the conventional '' Y-T coupling '' model for response regulators, phosphorylation induces the displacement of the conserved Thr87 residue through hydrogen-bond formation, which in turn makes it sterically possible for Tyr106 to isomerize from a solvent exposed configuration to a buried rotameric state. More than 160 unbiased activation trajectories show, however, that the rotation of Tyr106 does not rely on the displacement of Thr87 per se. Free energy calculations reveal that the Tyr106 rotation is a low-barrier process in the absence of the Thr87-phosphate hydrogen bond, although the rotation is stabilized by the formation of this interaction. The simulations also find that structural change in the beta 4-alpha 4 loop does not gate the Tyr106 rotation as suggested previously; rather, the rotation of Tyr106 stabilizes the activated configuration of this loop. The computational strategy used and mechanistic insights obtained have an impact on the study of signaling proteins and allosteric systems in general.