Adenosine receptors are G protein-coupled receptors that sense extracellular adenosine to transmit intracellular signals. One of the four adenosine receptor subtypes, the adenosine A_2A receptor (A_2AR), has an exceptionally long intracellular C terminus (A_2AR-ct) that mediates interactions with a large array of proteins, including calmodulin and α-actinin. Here, we aimed to ascertain the α-actinin 1/calmodulin interplay whilst binding to A_2AR and the role of Ca^2 + in this process. First, we studied the A_2AR-α-actinin 1 interaction by means of native polyacrylamide gel electrophoresis, isothermal titration calorimetry, and surface plasmon resonance, using purified recombinant proteins. α-Actinin 1 binds the A_2AR-ct through its distal calmodulin-like domain in a Ca^2 +-independent manner with a dissociation constant of 5–12 μM, thus showing an ~ 100 times lower affinity compared to the A_2AR-calmodulin/Ca^2 + complex. Importantly, calmodulin displaced α-actinin 1 from the A_2AR-ct in a Ca^2 +-dependent fashion, disrupting the A_2AR-α-actinin 1 complex. Finally, we assessed the impact of Ca^2 + on A_2AR internalization in living cells, a function operated by the A_2AR-α-actinin 1 complex. Interestingly, while Ca^2 + influx did not affect constitutive A_2AR endocytosis, it abolished agonist-dependent internalization. In addition, we demonstrated that the A_2AR/α-actinin interaction plays a pivotal role in receptor internalization and function. Overall, our results suggest that the interplay of A_2AR with calmodulin and α-actinin 1 is fine-tuned by Ca^2 +, a fact that might power agonist-mediated receptor internalization and function.