Copyright © 2019 The Authors, some rights reserved; Ras-related C3 botulinum toxin substrate 1 (Rac1) functions as a molecular switch by cycling between an inactive guanosine diphosphate (GDP)–bound state and an active guanosine triphosphate (GTP)–bound state. An oncogenic mutant of Rac1, an N92I mutant, strongly promotes cell proliferation and subsequent oncogenic activities by facilitating the intrinsic GDP dissociation in the inactive GDP-bound state. Here, we used solution nuclear magnetic resonance spectroscopy to investigate the activation mechanism of the N92I mutant. We found that the static structure of the GDP binding site is not markedly perturbed by the mutation, but the overall conformational stability decreases in the N92I mutant, which then facilitates GDP dissociation by lowering the activation energy for the dissociation reaction. On the basis of these results, we proposed the activation mechanism of the N92I mutant, in which the decreased conformational stability plays important roles in its activation process.