The TWIK-related spinal cord K channel (TRESK, K18.1) is a K channel contributing to the maintenance of membrane potentials in various cells. Recently, physiological TRESK function was identified as a key player in T-cell differentiation rendering the channel a new pharmacological target for treatment of autoimmune diseases. The channel activator cloxyquin represents a promising lead compound for the development of a new class of immunomodulators. Identification of cloxyquin binding site and characterization of the molecular activation mechanism can foster the future drug development. Here, we identify the cloxyquin binding site at the M2/M4 interface by mutational scan and analyze the molecular mechanism of action by protein modeling as well as in silico and in vitro electrophysiology using different permeating ion species (K / Rb). In combination with kinetic analyses of channel inactivation, our results suggest that cloxyquin allosterically stabilizes the inner selectivity filter facilitating the conduction process subsequently activating hTRESK.