SUMOylation, covalent conjugation of small ubiquitin-related modifier (SUMO), has been emerging as a critical posttranslational modification of developmental transcription factors, as well as key regulators in the adult heart. Identifying the SUMOylated targets within cardiac transcription factors will facilitate to unravel the roles of SUMOylation in heart development and disease. Here, we show that Gata6, an essential cardiac transcription factor, can be modified by SUMO in vivo. Mutation of potential SUMOylation sites reveals that a lysine residue at amino acid position 12 of Gata6 serves as the major attachment site for SUMO. Pias1, as an E3 SUMO ligase, preferentially enhances the conjugation of SUMO1 to Gata6 through its RING finger domain. Functional analyses with SUMOylation-deficient mutant indicate that SUMOylation does not affect the subcellular localization but instead represses Gata6 transcriptional activity. Our data suggest that posttranslational modification of Gata6 by SUMO conjugation provides a novel mechanism to regulate Gata6 activity.