The voltage-gated sodium channel [pore-forming subunit of the neuronal voltage-gated sodium channel (Na_V1.6)] has recently been found in cardiac myocytes. Emerging studies indicate a role for Na_V1.6 in ionic homeostasis as well as arrhythmogenesis. Little is known about the spatial organization of these channels in cardiac muscle, mainly due to the lack of high-fidelity antibodies. Therefore, we developed and rigorously validated a novel rabbit polyclonal Na_V1.6 antibody and undertook super-resolution microscopy studies of Na_V1.6 localization in cardiac muscle. We developed and validated a novel rabbit polyclonal antibody against a C-terminal epitope on the neuronal sodium channel 1.6 (Na_V1.6). Raw sera showed high affinity in immuno-fluorescence studies, which was improved with affinity purification. The antibody was rigorously validated for specificity via multiple approaches. Lastly, we used this antibody in proximity ligation assay (PLA) and super-resolution STochastic Optical Reconstruction Microscopy (STORM) studies, which revealed enrichment of Na_V1.6 in close proximity to ryanodine receptor (RyR2), a key calcium (Ca²⁺) cycling protein, in cardiac myocytes. In summary, our novel Na_V1.6 antibody demonstrates high degrees of specificity and fidelity in multiple preparations. It enabled multimodal microscopic studies and revealed that over half of the Na_V1.6 channels in cardiac myocytes are located within 100 nm of ryanodine receptor Ca²⁺ release channels.