Melatonin receptors (MT₁ and MT₂) transduce inhibitory signaling by melatonin (N-acetyl-5-methoxytryptamine), which is associated with sleep induction and circadian rhythm modulation. Although recently reported crystal structures of ligand-bound MT₁ and MT₂ elucidated the basis of ligand entry and recognition, the ligand-induced MT₁ rearrangement leading to G_i-coupling remains unclear. Here we report a cryo-EM structure of the human MT₁-G_i signaling complex at 3.3 Å resolution, revealing melatonin-induced conformational changes propagated to the G-protein-coupling interface during activation. In contrast to other G_i-coupled receptors, MT₁ exhibits a large outward movement of TM6, which is considered a specific feature of G_s-coupled receptors. Structural comparison of G_i and G_s complexes demonstrated conformational diversity of the C-terminal entry of the G_i protein, suggesting loose and variable interactions at the end of the α5 helix of G_i protein. These notions, together with our biochemical and computational analyses, highlight variable binding modes of Gα_i and provide the basis for the selectivity of G-protein signaling.