Significance Genome-wide association studies have identified common noncoding variants associated with ECG parameters in the TBX3 locus, suggesting that a modest change in TBX3/Tbx3 expression level affects heart function. We characterized the morphology, physiology, and gene expression profile of the atrioventricular conduction system in a Tbx3 haploinsufficient mouse model. We identified Tbx3-dependent regulatory DNA elements active in the atrioventricular conduction system and validated the functionality of these elements in vivo. Using genome editing, we deleted a regulatory DNA element from the mouse genome and show it drives expression of Cacna1g in the cardiac conduction system. Our study unveils a Tbx3-dependent gene regulatory network in the AV conduction system that contributes to heart electrophysiology.