Impaired fasting glucose (IFG) and impaired glucose tolerance (IGT) are high-risk factors of diabetes development and may be caused by defective insulin secretion in pancreatic beta-cells. Glucose-stimulated insulin secretion is mediated by voltage-gated Ca²⁺ (Ca_V) channels in which the gamma-4 subunit (Ca_Vγ4) is required for the beta-cell to maintain its differentiated state. We here aim to explore the involvement of Ca_Vγ4 in controlling glucose homeostasis by employing the Ca_Vγ4^−/− mice to study in vivo glucose-metabolism-related phenotypes and glucose-stimulated insulin secretion, and to investigate the underlying mechanisms. We show that Ca_Vγ4^−/− mice exhibit perturbed glucose homeostasis, including IFG and IGT. Glucose-stimulated insulin secretion is blunted in Ca_Vγ4^−/− mouse islets. Remarkably, Ca_Vγ4 deletion results in reduced expression of the transcription factor essential for beta-cell maturation, MafA, on both mRNA and protein levels in islets from human donors and Ca_Vγ4^−/− mice, as well as in INS-1 832/13 cells. Moreover, we prove that CaMKII is responsible for mediating this regulatory pathway linked between Ca_Vγ4 and MafA, which is further confirmed by human islet RNA-seq data. We demonstrate that Ca_Vγ4 is a key player in preserving normal blood glucose homeostasis, which sheds light on Ca_Vγ4 as a novel target for the treatment of prediabetes through correcting the impaired metabolic status.