Modification to the structure of glucagon has provided a number of glucagon receptor antagonists with possible therapeutic application for diabetes. These novel peptide analogs include desHis1Pro4Glu9-glucagon and desHis1Pro4Glu9(Lys30PAL)-glucagon. This study has evaluated the metabolic benefits of once daily administration of desHis1Pro4Glu9-glucagon and desHis1Pro4Glu9(Lys30PAL)-glucagon in high fat (45%) fed mice for 15 days. Administration of desHis1Pro4Glu9-glucagon and desHis1Pro4Glu9(Lys30PAL)-glucagon had no significant effect on body weight, food intake or circulating glucose concentrations during the treatment period. However, both peptides significantly (P < 0.05 to P < 0.01) reduced circulating plasma insulin concentrations from day 6 onwards. Oral glucose tolerance and insulin sensitivity, as assessed by exogenous insulin administration, were significantly (P < 0.01 to P < 0.001) improved by both desHis1Pro4Glu9-glucagon and desHis1Pro4Glu9(Lys30PAL)-glucagon. These metabolic benefits were accompanied by significantly (P < 0.01) increased pancreatic insulin stores. No significant differences in blood triacylglycerol or cholesterol levels were noted with desHis1Pro4Glu9-glucagon, however desHis1Pro4Glu9(Lys30PAL)-glucagon treatment significantly (P < 0.01) increased HDL-cholesterol levels. Glucagon-mediated elevations of glucose and insulin were effectively (P < 0.01 to P < 0.001) annulled in both treatment groups on day 15. Interestingly, glucose levels during an intraperitoneal glucose tolerance test were not altered by either desHis1Pro4Glu9-glucagon or desHis1Pro4Glu9(Lys30PAL)-glucagon treatment. These data provide further evidence that glucagon antagonism could provide an effective means of treating T2DM.