GPRC6A is proposed to regulate energy metabolism in mice, but in humans a KGKY polymorphism in the third intracellular loop (ICL3) is proposed to result in intracellular retention and loss-of-function. To test physiological importance of this human polymorphism in vivo, we performed targeted genomic humanization of mice by using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR associated protein 9) system to replace the RKLP sequence in the ICL3 of the GPRC6A mouse gene with the uniquely human KGKY sequence to create Gprc6a-KGKY-knockin mice. Knock-in of a human KGKY sequence resulted in a reduction in basal blood glucose levels and increased circulating serum insulin and FGF-21 concentrations. Gprc6a-KGKY-knockin mice demonstrated improved glucose tolerance, despite impaired insulin sensitivity and enhanced pyruvate-mediated gluconeogenesis. Liver transcriptome analysis of Gprc6a-KGKY-knockin mice identified alterations in glucose, glycogen and fat metabolism pathways. Thus, the uniquely human GPRC6A-KGKY variant appears to be a gain-of-function polymorphism that positively regulates energy metabolism in mice.