Glucose homeostasis is a complex indispensable process and its dysregulation causes hyperglycaemia and type 2 diabetes mellitus. Glucokinase (GK) takes a central role in these pathways and thus is rate-limiting for glucose-stimulated insulin secretion (GSIS) from pancreatic islets. Several reports have described the transcriptional regulation of Gck mRNA, while its post-transcriptional mechanisms of regulation, especially those involving microRNAs (miR), are poorly understood. In this study, we investigated the role of miR-206 as a post-transcriptional regulator of Gck. In addition, we examined the effects of miR-206 on glucose tolerance, GSIS, and gene expression in control and germ line miR-206 knock-out (KO) mice fed either with chow or high-fat diet (HFD). MiR-206 was found in Gck-expressing tissues and was differentially altered in response to HFD-feeding. Pancreatic islets showed the most profound induction in the expression of miR-206 in response to HFD. Chow- and HFD-fed miR-206 KO mice have improved glucose tolerance and GSIS but unaltered insulin sensitivity. In silico analysis of Gck mRNA revealed a conserved 8-mer miR-206 binding site. Hence, the predicted regulation of Gck by miR-206 was confirmed in reporter and GK activity assays. Concomitant with increased GK activity, miR-206KO mice had elevated liver glycogen content and plasma lactate concentrations. Our findings revealed a novel mechanism of post-transcriptional regulation of Gck by miR-206 and underline the crucial role of pancreatic islet miR-206 in the regulation of whole-body glucose homeostasis in a murine model that mimics the metabolic syndrome.