Insulin and its derivatives are life-saving drugs for people with type 1 diabetes (T1D) and some type 2 diabetes (T2D). Insulin is a peptide hormone composed of two peptide chains, the A chain (21 amino acids with an intra-chain disulfide bond) and the B chain (30 amino acids), linked together by two disulfide bonds. All U.S. Food and Drug Administration (FDA)-approved insulin analogs to date have modifications on the C-terminal region of the B chain to alter pharmacokinetic properties of insulin for improved glucose controls. Additional insulin modifications on the C-terminal region of the B chain were reported to achieve glucose responsiveness, aggregation resistance, and insulin receptor (IR) isoform selectivity. These studies demonstrate that the C-terminal region of the B chain is not critical to insulin bioactivity and can tolerate additional conjugates to some degree, which is supported by the fact that des-B26-30 insulinamide (deletion of B26-30) is fully active. Although modification of the Cterminal region of the B chain has been proved its versatility, compatibility, and reliability for extending the functional scope of insulin, facile methods to fully explore this region is still limited: 1) Acylation on LysB29 has a limited chemical reaction scope. 2) Trypsin-mediated ligation using desoctapeptide (B23-30) insulin (DOI) suffers from low conversion rates (Figure ). Our group recently reported sortase A-mediated ligation with a high conversion rate (Figure ). However, it can only conjugate peptide substrates after the C-terminus of the B chain. A new way to fully explore the functional scope of the B26-30 region of insulin is highly needed. Omniligase-1, a variant of subtiligase, has a broad substrate peptide sequence scope. A peptide ester is commonly used as a non-prime side substrate for omniligase-1, and ligated to the N-terminus of another prime-side peptide. Omniligase-1 first uses the thiol group of the catalytic cysteine to activate the ester bond of the substrate peptide ester through transthioesterification. Omniligase-1 then recruits the prime-side peptide and uses its N-terminus to release the enzymeconjugated peptide through amidation (Scheme S1). Furthermore, omniligase-1 recognizes the P1-P4 residues of the nonprime side peptide and the P1'-P2' residues of the prime-side peptide with various ligation efficiencies. We hypothesized that insulin ester with a truncated C-terminal region of the B chain could be ligated with the N-terminus of another peptide by omniligase-1 (Figure ) to make insulin in the same manner as sortase A-mediated ligation. If it works, the omniligase-1-mediated ligation will allow modifications on the B26-30 region of insulin with high conversion rates. Herein, we described a synthetic method of insulin through omnniligase-1 ligation between B24-25 with high conversion rates. We further demonstrated that this omniligase-1 approach can be used for phage surface ligation. This work showed the first example of omniligase-1-based phage modification and paved the way of future applications using phage display libraries for ligand selections.