The function, regulation, and molecular structure of the cardiac Na⁺/Ca²⁺ exchangers (NCXs) vary significantly among vertebrates. We previously reported that -adrenergic suppression of amphibian cardiac NCX1.1 is associated with specific molecular motifs. Here we investigated the bimodal, cAMP-dependent regulation of spiny dogfish shark (Squalus acanthias) cardiac NCX, exploring the effects of molecular structure, host cell environment, and ionic milieu. The shark cardiac NCX sequence (DQ 068478) revealed two novel proline/alanine-rich AA-insertions. Wild type and mutant shark NCXs were cloned and expressed in mammalian cells (HEK 293 and FlpIn 293), where their activities were measured as Ni²⁺-sensitive Ca²⁺-fluxes (Fluo-4) and membrane currents (I_NaCa) evoked by changes in [Na⁺]_o and/or membrane potential (Vm). Regardless of Ca²⁺ buffering, -adrenergic stimulation of cloned wild type shark NCX consistently produced bimodal regulation (defined as differential regulation of Ca²⁺-efflux and influx pathways), with suppression of the Ca²⁺-influx mode and either no change or enhancement of the Ca²⁺-efflux mode, closely resembling results from parallel experiments with native shark cardiomyocytes. In contrast, mutant shark NCX, with deletion of the novel region # 2-insertion, produced equal suppression of the inward and outward currents and Ca²⁺ fluxes, thereby abolishing the bimodal nature of the regulation. Control experiments with non-transfected and dog cardiac NCX expressing cells showed no cAMP regulation. We conclude that bimodal -adrenergic regulation is retained in cloned shark NCX and is dependent on the shark's unique molecular motifs.