Cardiac chamber specification is driven by complex morphogenetic movements orchestrated by multiple signaling pathways. The SMyHC3 gene has been shown to play a significant role in determining atrial identity. It has been previously demonstrated that expression of the SMyHC3 quail gene is repressed in the ventricles via a VDRE/RARE element. However, the sequence driving its atrial specific expression remains elusive. Here, we tested the hypothesis that a new dual cis-regulatory element (the complex Nuclear Receptor Element, cNRE) found in the SMyHC3 promoter holds the information that governs SMyHC3 expression in atria and repression in the ventricle. By means of calorimetry and binding assays, we found that cNRE is composed of three tandemly arrayed hexads harboring binding sites for multiple nuclear receptors and it is the molecular switch determining the cell identity between atria or ventricle. The chicken ovalbumin promoter-transcription factor II (COUPTF-II), known to be essential and sufficient in driving the atrial cell fate is one of its ligands. Proteomic studies showed that COUPTF-II binds to the Androgen Receptor (AR) in order to activate the SMyHC3 promoter, confirmed by transactivation assays performed in HEK 293T cells. Transgenesis assays in mice and zebrafish have shown that the cNRE holds the information required to atrial activation and ventricular repression of the SMyHC3 promoter, and this regulatory element is sufficient to switch the ventricular specific expression towards an atrial. Taking together, these findings evidenced a novel conserved regulatory mechanism governing the atrial specific expression of the SMyHC3 gene and unveil new roles for known transcription factors in cell fate and cardiac chamber specification.