The involvement of a gut-bone axis in controlling bone physiology has been long suspected, although the exact mechanisms are unclear. We explored whether glucose-dependent insulinotropic polypeptide (GIP)-producing enteroendocrine K cells were involved in this process. The bone phenotype of transgenic mouse models lacking GIP secretion (GIP-GFP-KI) or enteroendocrine K cells (GIP-DT) was investigated. Mice deficient in GIP secretion exhibited lower bone strength, trabecular bone mass, trabecular number, and cortical thickness, notably due to higher bone resorption. Alterations of microstructure, modifications of bone compositional parameters, represented by lower collagen cross-linking, were also apparent. None of these alterations were observed in GIP-DT mice lacking enteroendocrine K cells, suggesting that another K-cell secretory product acts to counteract GIP action. To assess this, stable analogues of the known K-cell peptide hormones, xenin and GIP, were administered to mature NIH Swiss male mice. Both were capable of modulating bone strength mostly by altering bone microstructure, bone gene expression, and bone compositional parameters. However, the two molecules exhibited opposite actions on bone physiology, with evidence that xenin effects are mediated indirectly, possibly via neural networks. Our data highlight a previously unknown interaction between GIP and xenin, which both moderate gut-bone connectivity. (c) 2020 American Society for Bone and Mineral Research.