Defective L‐type Ca²⁺ channel (I_CaL) regulation is one major cause for contractile dysfunction in the heart. The I_CaL is enhanced by sympathetic nervous stimulation: via the activation of β‐adrenergic receptors, PKA phosphorylates the α1C(Ca_V1.2)‐ and β2‐channel subunits and ahnak, an associated 5643‐amino acid (aa) protein. In this study, we examined the role of a naturally occurring, genetic variant Ile5236Thr‐ahnak on I_CaL. Binding experiments with ahnak fragments (wild‐type, Ile5236Thr mutated) and patch clamp recordings revealed that Ile5236Thr‐ahnak critically affected both β2 subunit interaction and I_CaL regulation. Binding affinity between ahnak‐C1 (aa 4646‐5288) and β2 subunit decreased by ≈50% after PKA phosphorylation or in the presence of Ile5236Thr‐ahnak peptide. On native cardiomyocytes, intracellular application of this mutated ahnak peptide mimicked the PKA‐effects on I_CaL increasing the amplitude by ≈60% and slowing its inactivation together with a leftward shift of its voltage dependency. Both mutated Ile5236Thr‐peptide and Ile5236Thr‐fragment (aa 5215‐5288) prevented specifically the further up‐regulation of I_CaL by isoprenaline. Hence, we suggest the ahnak‐C1 domain serves as physiological brake on I_CaL. Relief from this inhibition is proposed as common pathway used by sympathetic signaling and Ile5236Thr‐ahnak fragments to increase I_CaL. This genetic ahnak variant might cause individual differences in I_CaL regulation upon physiological challenges or therapeutic interventions.—Haase, H., Alvarez, J., Petzhold, D., Doller, A., Behlke, J., Erdmann, J., Hetzer, R., Regitz‐Zagrosek, V., Vassort, G., Morano, I. Ahnak is critical for cardiac Ca(v)1.2 calcium channel function and its β‐adrenergic regulation. FASEB J. 19, 1969–1977 (2005)