Exchange protein directly activated by cAMP (EPAC1) mediates the intracellular functions of a critical stress-response second messenger, cAMP. Herein, we report that EPAC1 is a cellular substrate of protein SUMOylation, a prevalent stress-response posttranslational modification. Site-specific mapping of SUMOylation by mass spectrometer leads to identifying K561 as a primary SUMOylation site in EPAC1. Sequence and site-directed mutagenesis analyses reveal a functional SUMO-interacting motif required for cellular SUMOylation of EPAC1. SUMO modification of EPAC1 mediates its heat shock-induced Rap1/2 activation in a cAMP-independent manner. Structural modeling and molecular dynamics simulation studies demonstrate that SUMO substituent on K561 of EPAC1 promotes Rap1 interaction by increasing the buried surface area between the SUMOylated receptor and its effector. Our studies identify a functional SUMOylation site in EPAC1 and unveil a novel mechanism in which SUMOylation of EPAC1 leads to its autonomous activation. The findings of SUMOylation-mediated activation of EPAC1 not only provide new insights into our understanding of cellular regulation of EPAC1 but also will open up a new field of experimentation concerning the cross-talk between cAMP/EPAC1 signaling and protein SUMOylation, two major cellular stress response pathways, during cellular homeostasis.