Voltage-gated K(+) channels (Kv) are responsible for repolarizing excitable cells and can be heavily glycosylated. Cardiac Kv activity is indispensable where even minimal reductions in function can extend action potential duration, prolong QT intervals, and ultimately contribute to life-threatening arrhythmias. Diseases such as congenital disorders of glycosylation often cause significant cardiac phenotypes that can include arrhythmias. Here we investigated the impact of reduced sialylation on ventricular repolarization through gene deletion of the sialyltransferase ST3Gal4. ST3Gal4-deficient mice (ST3Gal4(-/-)) had prolonged QT intervals with a concomitant increase in ventricular action potential duration. Ventricular apex myocytes isolated from ST3Gal4(-/-) mice demonstrated depolarizing shifts in activation gating of the transient outward (Ito) and delayed rectifier (IKslow) components of K(+) current with no change in maximum current densities. Consistently, similar protein expression levels of the three Kv isoforms responsible for Ito and IKslow were measured for ST3Gal4(-/-) versus controls. However, novel non-enzymatic sialic acid labeling indicated a reduction in sialylation of ST3Gal4(-/-) ventricular Kv4.2 and Kv1.5, which contribute to Ito and IKslow, respectively. Thus, we describe here a novel form of regulating cardiac function through the activities of a specific glycogene product. Namely, reduced ST3Gal4 activity leads to a loss of isoform-specific Kv sialylation and function, thereby limiting Kv activity during the action potential and decreasing repolarization rate, which likely contributes to prolonged ventricular repolarization. These studies elucidate a novel role for individual glycogene products in contributing to a complex network of cardiac regulation under normal and pathologic conditions.