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The atrioventricular node (AVN) coordinates the timing of atrial and ventricular contraction to optimize cardiac performance. To study this critical function using mouse genetics, however, new reagents are needed that allow AVN-specific manipulation. Here we describe a novel Gjd3-CreEGFP mouse line that successfully recombines floxed alleles within the AVN beginning at E12.5. These mice have been engineered to express CreEGFP under the control of endogenous Gjd3 regulatory elements without perturbing native protein expression. Detailed histological analysis of Gjd3-CreEGFP mice reveals specific labeling of AVN cardiomyocytes and a subset of cardiac endothelial cells. Importantly, we show that Gjd3-CreEGFP mice have preserved cardiac mechanical and electrical function. In one application of our newly described mouse line, we provide a three-dimensional (3D) view of the AVN using tissue clearing combined with confocal microscopy. With this 3D model as a reference, we identify specific AVN sub-structures based on marker staining characteristics. In addition, we use our Gjd3-CreEGFP mice to guide microdissection of the AVN and construction of a single-cell atlas. Thus, our results establish a new transgenic tool for AVN-specific recombination, provide an updated model of AVN morphology, and describe a roadmap for exploring AVN cellular heterogeneity.