Insulin is one of the most marketed therapeutic proteins worldwide. However, its formulation suffers from fibrillation, which affects the long-term storage limiting the development of novel devices for sustained delivery including portable infusion devices. We have investigated the effect of physical PEGylation on structural and colloidal stability of insulin by using 2 PEGylating agents terminating with polycyclic hydrophobic moieties, cholane and cholesterol: mPEG-cholane and mPEG-cholesterol, respectively. Microcalorimetric analyses showed that mPEG-cholane and mPEG-cholesterol efficiently bind insulin with binding constants (Ka) of 3.98 10 and 1.14 10 M, respectively. At room temperature, the 2 PEGylating agents yielded comparable structural stabilization of α-helix conformation and decreased dimerization of insulin. However, melting studies showed that mPEG-cholesterol has superior stabilizing effect of the protein conformation than mPEG-cholane. Furthermore, the fibrillation study showed that at a 1:1 and 1:5 insulin/polymer molar ratios, mPEG-cholesterol delays insulin fibrillation 40% and 26% more efficiently, respectively, as compared to mPEG-cholane which was confirmed by transmission electron microscopy imaging. Insulin was released from the mPEG-cholane and mPEG-cholesterol assemblies with comparable kinetic profiles. The physical PEGylation has a beneficial effect on the stabilization and shielding of the insulin structure into the monomeric form, which is not prone to fibrillation and aggregation.