Exchange proteins directly activated by cAMP (EPACs) are important allosteric regulators of cAMP-mediated signal transduction pathways. To understand the molecular mechanism of EPAC activation, we have combined site-directed mutagenesis, X-ray crystallography, and peptide amide hydrogen/deuterium exchange mass spectrometry (DXMS) to probe the structural and conformational dynamics of EPAC2-F435G, a constitutively active EPAC2 mutant. Our study demonstrates that conformational dynamics plays a critical role in cAMP-induced EPAC activation. A glycine mutation at 435 position shifts the equilibrium of conformational dynamics towards the extended active conformation.