The principal bone mineralisation inhibitor ectonucleotide pyrophosphatase/phosphodiesterase-1 (NPP1) has recently emerged as a pathogenic factor predisposing to insulin resistance and type 2 diabetes mellitus. We have previously demonstrated that Enpp1-/- mice exhibit pronounced resistance to obesity and insulin resistance following chronic high-fat diet (HFD) feeding. We also demonstrated that Enpp1-/- mice present with significant ectopic mineralisation (e.g. of the vasculature) and a paradoxical hypomineralisation of the long bones. Here, we determined the impact of osteoblast-specific Enpp1 ablation on HFD-induced obesity and skeletal phenotype. Mice lacking NPP1 specifically in osteoblasts (Ob-KO) were generated by crossing Enpp1flox/flox and Osteocalcin-Cre mice. Male mice were reared on a control diet (6.2% fat) or HFD (58% fat) up to 16-weeks of age. Metabolic tests, micro-computed tomography of long bones, dissection, gait, and histological analysis were performed. For data of two groups, t-test analysis was conducted. For data of more than two groups, one-way analysis of variance (ANOVA) using a general linear model and incorporating Tukey pairwise comparisons was performed. Under control dietary conditions, Ob-KO mice exhibited increased bone mass, indicated by increased femoral bone volume/total volume (149%; P<0.01), and reduced trabecular spacing (84%; P<0.01) at 6-weeks of age. Adult Ob-KO mice exhibit increased muscle mass (13.3%; P<0.05), likely resultant of increased gait instability (increased base of support (115%; P<0.05), girdle support (166%; P<0.05), initial (117%; P<0.05) and terminal (128%; P<0.05) forelimb dual stance). Adult mice showed normal glucose and insulin tolerance tests (GTT/ITT) comparable between genotypes. No notable differences in body weight or gross anatomy were observed. Following chronic HFD-challenge, no significant differences in body weight gain, muscle or white fat mass were observed between genotypes. However, Ob-KO mice showed significantly increased liver (130.1%; P<0.05) and brown fat mass (122.2%; P<0.05). These Ob-KO mice showed decreased glucose and insulin tolerance (P<0.05) following GTT and ITT respectively. No significant difference in pancreatic islet size or number between genotype were observed. These data indicate that the protection against diabetes noted in Enpp1-/- mice is likely due to the actions of non-skeletal NPP1. Indeed, osteoblast-specific NPP1 deficiency drives an unexpected worsening of insulin sensitivity relative to control mice on HFD. Together, these data indicate that NPP1 inhibition at its major site of expression is metabolically detrimental. Further studies are required to establish the mechanisms through which NPP1 regulates its actions within and out-with bone.