Pancreatic β-cell expansion is a highly regulated metabolic adaptation to increased somatic demands, including obesity and pregnancy; adult β cells otherwise rarely proliferate. We previously showed that high-fat diet (HFD) feeding induces mouse β-cell proliferation in less than 1 wk in the absence of insulin resistance. Here we metabolically profiled tissues from a short-term HFD β-cell expansion mouse model to identify pathways and metabolite changes associated with β-cell proliferation. Mice fed HFD vs. chow diet (CD) showed a 14.3% increase in body weight after 7 days; β-cell proliferation increased 1.75-fold without insulin resistance. Plasma from 1-wk HFD-fed mice induced β-cell proliferation ex vivo. The plasma, as well as liver, skeletal muscle, and bone, were assessed by LC and GC mass-spectrometry for global metabolite changes. Of the 1,283 metabolites detected, 159 showed significant changes [false discovery rate (FDR) < 0.1]. The majority of changes were in liver and muscle. Pathway enrichment analysis revealed key metabolic changes in steroid synthesis and lipid metabolism, including free fatty acids and other bioactive lipids. Other important enrichments included changes in the citric acid cycle and 1-carbon metabolism pathways implicated in DNA methylation. Although the minority of changes were observed in bone and plasma (<20), increased p-cresol sulfate was increased >4 fold in plasma (the largest increase in all tissues), and pantothenate (vitamin B) decreased >2-fold. The results suggest that HFD-mediated β-cell expansion is associated with complex, global metabolite changes. The finding could be a significant insight into Type 2 diabetes pathogenesis and potential novel drug targets.