Abstract Weekly basal insulin injections may increase treatment adherence in subjects with diabetes and an appropriately engineered weekly basal insulin may reduce daily pharmacokinetic (PK)/pharmacodynamic (PD) fluctuations compared to currently available daily basal insulins. Therefore, a weekly insulin has the potential to not only ease the burden of insulin therapy, but also improve outcomes for subjects with diabetes in a real-world setting. Basal insulin Fc (BIF, LY3209590) is an insulin Fc-fusion protein in clinical testing as a once weekly treatment for type 1 and type 2 diabetes mellitus (T1DM, T2DM). BIF is comprised of a human single-chain insulin fused to a human IgG2 Fc domain through a peptide linker. The in vitro evaluation determined that BIF exhibited reduced insulin receptor (IR) potency with full agonism, selectivity against human insulin-like growth factor-1 receptor (hIGF-1R), and functional properties similar to native human insulin. The binding affinity of BIF for hIR isoform A, Ki = 25 nM (SEM = 4, n=10), and hIR isoform B, Ki = 26 nM (SEM = 4, n=10), was more than two orders of magnitude weaker than human insulin. BIF stimulated IR phosphorylation in cells with reduced potency, but full agonism, and showed a significantly faster hIR dephosphorylation profile than either human insulin or AspB10 insulin. BIF stimulated de novo lipogenesis in 3T3-L1 adipocytes and cell proliferation in SAOS-2 and H4IIE cells with at least a 70-fold reduction in potency compared to human insulin. BIF possessed markedly reduced binding and activation of hIGF-1R making definitive mitogenic measurements unattainable. In preclinical in vivo pharmacology studies using streptozotocin (STZ)-treated diabetic rats, a statistically significant decrease in blood glucose compared to vehicle-treated animals was seen 24 hours post-injection and persisted through 336 hours post-injection following a single subcutaneous administration (30 nmol/kg) of BIF. In STZ-treated rats, BIF reached a Tmax at 48 hours, possessed an apparent clearance rate of ~0.85 mL/hr/kg, and t1/2 of ~120 hrs. Collectively, these results demonstrate that BIF possesses selective IR agonism with a pharmacological profile similar to native insulin, however with a significantly reduced potency, and a significantly extended time action profile in preclinical animal models supporting once weekly testing in the clinic.