Background and purpose:Gabapentin is an effective anticonvulsant. The major physiological function of renal outer medullary potassium (ROMK1) channels is to maintain the resting membrane potential (RMP). We investigated the effect of gabapentin on ROMK1 channels and the mechanism involved.Experimental approach:Xenopusoocytes were injected with mRNA coding for wild‐type or mutant ROMK1 channels and giant inside‐out patch‐clamp recordings were performed.Key results:Gabapentin increased the activity of ROMK1 channels, concentration‐dependently and enhanced the activity of wild‐type and an intracellular pH (pHi)‐gating residue mutant (K80M) channels over a range of pHi. Gabapentin also increased activity of channels mutated at phosphatidylinositol 4,5‐bisphosphate (PIP2)‐binding sites (R188Q, R217A and K218A). However, gabapentin failed to enhance channel activity in the presence of protein kinase A (PKA) inhibitors and did not activate phosphorylation site mutants (S44A, S219A or S313A), mutants that mimicked the negative charge carried by a phosphate group bound to a serine (S44D, S219D or S313D), or a mutated channel with a positive charge (S219R). These findings show that gabapentin activates ROMK1 channels independently of the pHiand not via a PIP2‐dependent pathway. The effects of gabapentin on ROMK1 channels may be due to a PKA‐mediated phosphorylation‐induced conformational change, but not to charge–charge interactions.Conclusions and implications:ROMK1 channels are the main channels responsible for maintaining the RMP during cellular excitation. Gabapentin increased the activity of ROMK1 channels by a PKA‐dependent mechanism, reducing neuronal excitability, and this may play an important role in its antiepileptic effect.