Background: The maintenance of the body's Mg2+ balance is of great importance because of its involvement in numerous enzymatic systems and its intervention in neuromuscular excitability, protein synthesis, and nucleic acid stability. Recently, the transient receptor potential melastatin 6 (TRPM6) was identified as the gatekeeper of active Mg2+ transport and therefore plays a crucial role in the regulation of Mg2+ homeostasis. Remarkably, TRPM6 combines a Mg2+ channel with an α-kinase domain whose function remains elusive. Results: Here, we identify the receptor for activated C-kinase 1 (RACK1) as the first regulatory protein of TRPM6 that associates with the α-kinase domain. RACK1 and TRPM6 are both present in renal Mg2+-transporting distal convoluted tubules. We demonstrate that RACK1 inhibits channel activity in an α-kinase activity-dependent manner, whereas small interference (si) RNA-mediated knockdown of RACK1 increases the current. Moreover, threonine1851 in the α-kinase domain was identified as an autophosphorylation site of which the phosphorylation state is essential for the inhibitory effect of RACK1. Importantly, threonine1851 was crucial for the Mg2+ sensitivity of TRPM6 autophosphorylation and channel activity. TRPM6 channel activity was less sensitive to Mg2+ when RACK1 was knocked down by siRNA. Finally, activation of protein kinase C by phorbol 12-myristate 13-acetate-PMA prohibited the inhibitory effect of RACK1 on TRPM6 channel activity. Conclusions: We propose a unique mode of TRPM6 regulation in which the Mg2+ influx is controlled by RACK1 through its interaction with the α-kinase and the phosphorylation state of the threonine1851 residue.