Although meta-analyses of genome-wide association studies have identified >60 single nucleotide polymorphisms (SNPs) associated with type 2 diabetes and/or glycemic traits, there is little information on whether these variants also affect α-cell function. The aim of the current study was to evaluate the effects of glycemia-associated genetic loci on islet function in vivo and in vitro. We studied 43 SNPs in 4,654 normoglycemic participants from the Finnish population-based Prevalence, Prediction, and Prevention of Diabetes-Botnia (PPP-Botnia) Study. Islet function was assessed, in vivo, by measuring insulin and glucagon concentrations during oral glucose tolerance test, and, in vitro, by measuring glucose-stimulated insulin and glucagon secretion from human pancreatic islets. Carriers of risk variants in BCL11A, HHEX, ZBED3, HNF1A, IGF1, and NOTCH2 showed elevated whereas those in CRY2, IGF2BP2, TSPAN8, and KCNJ11 showed decreased fasting and/or 2-h glucagon concentrations in vivo. Variants in BCL11A, TSPAN8, and NOTCH2 affected glucagon secretion both in vivo and in vitro. The MTNR1B variant was a clear outlier in the relationship analysis between insulin secretion and action, as well as between insulin, glucose, and glucagon. Many of the genetic variants shown to be associated with type 2 diabetes or glycemic traits also exert pleiotropic in vivo and in vitro effects on islet function.