Rab3 GTPase activating protein 1 (Rab3gap1) hydrolyzes GTP on Rab3 to inactivate it and reinitiate the Rab3 cycle that regulates exocytic release of neuropeptides and hormones from neuroendocrine cells and atrial natriuretic peptide (ANP) secretion by cardiomyocytes. Cysteine palmitoylation of Rab3gap1 by the Golgi-localized S-acyltransferase zDHHC9 was recently shown to hinder ANP release by impairing Rab3gap1-mediated nucleotide cycling on Rab3a. Here we interrogated the cysteine residues of Rab3gap1 modified by palmitoylation and impacts on ANP secretion in cardiomyocytes. Although mutation of the previously identified cysteine-678 (Cys-678) site of Rab3gap1 alone was insufficient to elicit complete loss of Rab3gap1 palmitoylation in cardiomyocytes, combinatorial mutation of Cys-509, 510, 521, 522, and 678 (Rab3gap1