The two-pore domain K(+) channel, TRESK (TWIK-related spinal cord K(+) channel, KCNK18) is directly regulated by the calcium/calmodulin-dependent phosphatase calcineurin and 14-3-3 adaptor proteins. The calcium signal robustly activates the channel via calcineurin, whereas the anchoring of 14-3-3 interferes with the return of the current to the resting state after the activation in Xenopus oocytes. In the present study, we report that the phosphorylation of TRESK at two distinct regulatory regions, the 14-3-3 binding site (Ser-264) and the cluster of three adjacent serine residues (Ser-274, Ser-276, and Ser-279), are responsible for channel inhibition. The phosphorylation of Ser-264 by protein kinase A accelerated the return of the current of S276E mutant TRESK to the resting state after the calcineurin-dependent activation. In the presence of 14-3-3, the basal current of the S276E mutant was reduced, and its calcineurin-dependent activation was augmented, suggesting that the direct binding of the adaptor protein to TRESK contributed to the basal inhibition of the channel under resting conditions. Unexpectedly, we found that 14-3-3 impeded the recovery of the current of S264E mutant TRESK to the resting state after the calcineurin-dependent activation, despite of the mutated 14-3-3 binding site. This suggests that 14-3-3 inhibited the kinase phosphorylating the regulatory cluster of Ser-274, Ser-276, and Ser-279, independently of the direct interaction between TRESK and 14-3-3. In conclusion, two distinct inhibitory kinase pathways converge on TRESK, and their effect on the calcineurin-dependent regulation is differentially modulated by the functional availability of 14-3-3.