Defatted soybean flour (DSF) can sorb and concentrate blueberry anthocyanins and other polyphenols, but not sugars. In this study blueberry polyphenol-enriched DSF (BB–DSF) or DSF were incorporated into very high fat diet (VHFD) formulations and provided ad libitum to obese and hyperglycemic C57BL/6 mice for 13 weeks to investigate anti-diabetic effects. Compared to the VHFD containing DSF, the diet supplemented with BB–DSF reduced weight gain by 5.6%, improved glucose tolerance, and lowered fasting blood glucose levels in mice within 7 weeks of intervention. Serum cholesterol of mice consuming the BB–DSF-supplemented diet was 13.2% lower than mice on the diet containing DSF. Compounds were eluted from DSF and BB–DSF for in vitro assays of glucose production and uptake. Compared to untreated control, doses of BB–DSF eluate containing 0.05–10μg/μL of blueberry anthocyanins significantly reduced glucose production by 24–74% in H4IIE rat hepatocytes, but did not increase glucose uptake in L6 myotubes. The results indicate that delivery of blueberry polyphenols stabilized in a high-protein food matrix may be useful for the dietary management of pre-diabetes and/or diabetes.