The beetle Oulema oryzae Kuwayama (Coleoptera: Chrysomelidae), an important pest of rice, has developed fipronil resistance in Japan. Molecular cloning and sequence analysis of O. oryzae RDL γ-aminobutyric acid (GABA) receptor subunit (OO-RDL) genes from fipronil-susceptible and -resistant O. oryzae identified the A2′S mutation (index number for the M2 membrane-spanning region). To investigate the effect of the A2′S mutation on fipronil resistance, we stably expressed the wild-type and mutant OO-RDL homomers in Drosophila Mel-2 cells. A membrane potential assay exhibited that the IC₅₀ values of fipronil for inhibition of the response to EC₈₀ GABA of the wild-type and A2′S mutant OO-RDL homomers were 0.09 µM and 0.11 µM, respectively. However, the IC₅₀ values of fipronil for inhibition of the response to EC₉₅ GABA of the wild-type and A2′S mutant OO-RDL homomers were 0.11 µM and ≈5 µM, respectively. These results suggest that the GABA concentration is an important factor affecting fipronil resistance in O. oryzae carrying the A2′S mutation in OO-RDL.