G-protein-gated inwardly rectifying K⁺ (GIRK1~4) channels play important roles in various organs. Mutations of GIRK channels are relevant to some diseases, such as Keppen-Lubinsky syndrome. These pathological mutations induce loss of K⁺ selectivity and thereby triggering disorders of cell functions. To clarify the mechanisms underlying the abnormal ion selectivity, we examined the ion selectivity profile of several GIRK2 mutants using various size of cations. By electrophysiological analysis, we observed that G156S in the selectivity filter (SF) has high Li⁺ and Na⁺ selectivity, while L173R in the transmembrane domain has high Rb⁺ and Cs⁺ selectivity. S148F and T151A in the pore helix also show abnormal ion selectivity. Applications of SF pathway blockers, Ba²⁺ or TPN-Q, change the ion selectivity of G156S and S148F but not those of L173R and T151A, suggesting that G156S and S148F may possess a second ion permeation pathway besides the SF pathway and that L173R and T151A may possess only a single ion pathway. By single-channel recordings of G156S, we observed two types of events that one ascribable to a TPN-Q-sensitive K⁺ current and the second a TPN-Q-resistant Li⁺ current. These results support that a novel Li⁺-permeable and blocker-resistant pathway exists in GIRK2 G156S in addition to the SF pathway.