GIRK (Kir3) channels are activated by neurotransmitters coupled to G proteins, via a direct binding of Gβγ. The role of Gα subunits in GIRK gating is elusive. Here we demonstrate that Gαi is not only a donor of Gβγ but also regulates GIRK gating. When overexpressed in Xenopus oocytes, GIRK channels show excessive basal activity and poor activation by agonist or Gβγ. Coexpression of Gαi3 or Gαi1 restores the correct gating parameters. Gαi acts neither as a pure Gβγ scavenger nor as an allosteric cofactor for Gβγ. It inhibits only the basal activity without interfering with Gβγ-induced response. Thus, GIRK is regulated, in distinct ways, by both arms of the G protein. Gαi probably acts in its GDP bound form, alone or as a part of Gαβγ heterotrimer.