TWIK1 is a 2-P-domain potassium channel which is mainly expressed in brain and kidney but also in other tissues like pancreatic islets. We investigated the possible role of TWIK1 for insulin secretion using in vivo and in vitro techniques. In murine pancreatic islets, strong TWIK1 immuno-staining was observed in insulin-producing cells. To test the relevance of TWIK1 for insulin secretion, we performed glucose tolerance tests: Male TWIK1 -/- mice exhibited normal peak values but a faster decrease of blood glucose after the peak. This was paralleled by higher insulin and C-peptide concentrations. Similar results were obtained from insulin secretion assays on isolated pancreatic islets. Additionally, blood glucose of -/- mice was lower and insulin and C-peptide concentrations were higher after fructose- rich diet. Interestingly, fura2 measurements on isolated beta cells showed not an enhanced but a slightly reduced Ca2+ response upon glucose (10 mM). Similarly, in patch-clamp experiments beta cells of -/- mice showed a reduced depolarization upon tolbutamide which is suggestive for an unexpected up-regulation of tolbutamide-insensitive K+ channels. Taken together, TWIK1 gene disruption increased insulin secretion. The underlying mechanism appears to be independent from Ca2+ and probably involves other pathways and/or hormones.