The subunits KCNQ1 and KCNE1 generate the slowly activating, delayed rectifier potassium current, I, that responds to sympathetic stimulation and is critical for human cardiac repolarization. The A-kinase anchoring protein Yotiao facilitates macromolecular complex formation between I and protein kinase A (PKA) to regulate phosphorylation of KCNQ1 and I currents following beta-adrenergic stimulation. We have previously shown that adenylyl cyclase Type 9 (AC9) is associated with a KCNQ1-Yotiao-PKA complex and facilitates isoproterenol-stimulated phosphorylation of KCNQ1 in an immortalized cell line. However, requirement for AC9 in sympathetic control of I in the heart was unknown. Using a transgenic mouse strain expressing the KCNQ1-KCNE1 subunits of I, we show that AC9 is the only adenylyl cyclase (AC) isoform associated with the KCNQ1-KCNE1-Yotiao complex in the heart. Deletion of AC9 resulted in the loss of isoproterenol-stimulated KCNQ1 phosphorylation in vivo, even though AC9 represents less than 3% of total cardiac AC activity. Importantly, a significant reduction of isoproterenol-stimulated I currents was also observed in adult cardiomyocytes from I-expressing AC9KO mice. AC9 and Yotiao co-localize with N-cadherin, a marker of intercalated disks and cell-cell junctions, in neonatal and adult cardiomyocytes, respectively. In conclusion, AC9 is necessary for sympathetic regulation of PKA phosphorylation of KCNQ1 in vivo and for functional regulation of I in adult cardiomyocytes.