Pancreatic β cell mass and function increase in conditions of enhanced insulin demand such as obesity. Failure to adapt leads to diabetes. The molecular mechanisms controlling this adaptive process are unclear. Fas is a death receptor involved in β cell apoptosis or proliferation, depending on the activity of the caspase-8 inhibitor FLIP. Here we show that the Fas pathway also regulates β cell secretory function. We observed impaired glucose tolerance in Fas-deficient mice due to a delayed and decreased insulin secretory pattern. Expression of PDX-1, a β cell-specific transcription factor regulating insulin gene expression and mitochondrial metabolism, was decreased in Fas-deficient β cells. As a consequence, insulin and ATP production were severely reduced and only partly compensated for by increased β cell mass. Up-regulation of FLIP enhanced NF-κB activity via NF-κB-inducing kinase and RelB. This led to increased PDX-1 and insulin production independent of changes in cell turnover. The results support a previously undescribed role for the Fas pathway in regulating insulin production and release.