Bifunctional fusion protein design has been widely utilized as a strategy to increase the efficacy of protein therapeutics. Previously, we proposed a novel application of the bifunctional fusion protein design through the introduction of proinsulin-transferrin (ProINS-Tf) fusion protein as a liver-specific protein prodrug to achieve a glucose-lowering effect in type 1 diabetic mice. In this report, we studied the binding characteristics of this activated fusion protein to the insulin receptor to elucidate its mechanism in eliciting insulin receptor-mediated signaling. We found that, with the assistance of the transferrin moiety binding to the transferrin receptor, the activated ProINS-Tf exhibited significantly higher binding affinity to the insulin receptor compared with the native insulin, resulting in a prolonged and stronger Akt phosphorylation. This enhanced induction by activated ProINS-Tf overcame insulin resistance in palmitate-treated HepG2 cells. ProINS-Tf also demonstrated a better glucose-lowering effect than native insulin, even with a much lower dose and less frequent injections, in non-obese diabetic mice with insulin resistance symptoms. The activated ProINS-Tf, serving as a bivalent protein molecule, could be a new insulin analog to overcome insulin resistance, which is associated with several diseases, including type 2 diabetes and non-alcoholic fatty liver disease. Insulin (INS) resistance is the major cause of the development of type 2 diabetes (T2D), and it is often referred as a state in which a higher than normal level of INS is required to achieve the normal response 1-3. INS resistance may result from alterations at different cellular levels, including insulin deficient signaling, inflammation, endoplasmic reticulum stress, and mitochondrial dysfunction 3. Type 2 diabetic patients often require intensive insulin treatment to maintain glycemic control, which leads to increased risk of hypoglycemia, weight gain, and further deterioration of INS resistance state 1,4. Previously, INS X10 with higher IR binding affinity have been studied as a rapid-acting INS analogue to treat type 2 diabetes 5. INS X10 demonstrated sustained effect in inducing IR-mediated signaling; however, the development of INS X10 was discontinued due to disproportionate increase in mitogenic activity and higher breast cancer incidence in the long-term rat studies 6. It is still a great challenge to develop novel INS analogues with enhanced binding affinity to the IR to overcome INS resistance safely and effectively. A proinsulin-transferrin (ProINS-Tf) fusion protein was previously developed as a novel long-acting and liver-targeted INS prodrug for treating type 1 diabetic (T1D) mice 7,8. Proinsulin (ProINS), as a precursor of insulin (INS), has a much lower binding affinity to INS receptor (IR) and the resultant biologic potency is only 1% or less relative to INS 9. Therefore, ProINS-Tf is initially inactive and requires a lag time to be activated before exhibiting activity in stimulating Akt phosphorylation in H4IIE ce...