There is growing evidence to suggest insulin signalling in bone tissue plays a critical role in the regulation of whole-body glucose and energy metabolism. However, a systems biology analysis to map in vivo signalling architecture has yet to be performed. Furthermore, whether this network of signalling is rewired during ageing and insulin-resistance is unknown. To this effect, we present the first mouse bone phosphoproteomics landscape from 8-week and 73-week old C57BL/6J mice following insulin or vehicle treatment. Tryptic bone lysates were labelled with Tandem Mass Tags and phosphopeptides enriched using titanium dioxide and analysed by multi-dimensional ultra-high-pressure liquid chromatography coupled to tandem mass spectrometry (2DLC-MS/MS), to compare in vivo age-related insulin signalling and to identify key regulators of the osteo-insulin pathway. The mouse bone phosphoproteome revealed 16,502 phosphosites that were mapped to 4528 bone phosphoproteins. Of these, we identified 4,661 novel phosphosites (~28% of the total phosphosites) that are associated with proteins involved in several regulatory mechanisms including the insulin signalling pathway. Comparison of insulin activated 8-week old bone to the control revealed 2,953 insulin-regulated phosphosites (q<0.05; Limma moderated t-test with Benjamini Hochberg FDR), including several substrates of insulin-responsive kinases such as AKT and mTOR. Furthermore, an age associated differential expression of 2,175 insulin-regulated phosphosites was observed in 8-week versus the 73-week old bone revealing dramatic rewiring and defects in insulin signalling (q<0.05; Limma ANOVA with Benjamini Hochberg FDR). We next utilised machine-learning based on kinase motif and expression profiles to predict substrates of Akt/S6K, mTOR and PKA kinase. Integration of these data with systems genetic analysis of human GWAS against bone mineral density enabled us to prioritise high confidence targets for functional validation. We further integrated these data with phosphosite evolutionary conservation analysis to prioritise functional phosphorylation sites with an emphasis on zebrafish. This enabled us to establish a CRISPR/Cas9 loss-of-function screen in zebrafish to identify novel phosphoproteins regulating bone formation and glucose metabolism. We present preliminary and ongoing advances in establishing high-throughput functional screening of zebrafish bone integrated with proteomics. We hope our functional analysis of the bone phosphoproteome will further enhance our understanding of the signalling mechanisms controlling bone biology and whole-body energy metabolism.