Protein motions are important to activity, but quantitative relationships between internal dynamics and function are not well understood. The Dbl homology (DH) domain of the proto-oncoprotein and guanine nucleotide exchange factor Vav1 is autoinhibited through interactions between its catalytic surface and a helix from an N-terminal acidic region. Phosphorylation of the helix relieves autoinhibition. Here we show by NMR spectroscopy that the autoinhibited DH domain exists in equilibrium between a ground state, where the active site is blocked by the inhibitory helix, and an excited state, where the helix is dissociated. Across a series of mutants that differentially sample these states, catalytic activity of the autoinhibited protein and its rate of phosphorylation are linearly dependent on the population of the excited state. Thus, internal dynamics are required for and control both basal activity and the rate of full activation of the autoinhibited DH domain.