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[Display omitted] Insulin treatment is currently considered to be the main strategy for controlling diabetes. Although the recombinant insulin formulation is relatively mature, we found that a batch of insulin formulation exhibited an unusual degradation rate in the stability experiment. The main purposes of this article are to identify the root cause for this phenomenon and characterize of chemical and physical degradation products. We compared the chemical and physical stability of two batches of insulin formulations prepared separately with simulated repeated use and freshly opened glycerol. The chemical stability of insulin was identified by liquid chromatography coupled with tandem mass spectrometry (LC- MS/MS). Micro-flow imaging (MFI), far-ultraviolet circular dichroism (Far-UV CD) and Thioflavin T (ThT) fluorescent assays were used to reveal protein aggregation and fibrosis. The chemical and physical stability of the insulin formulation with newly opened glycerol was much better than that with degraded glycerol, and both groups of formulations were extremely sensitive to light. The results indicated that the original batch insulin formulation with abnormal stability was indeed caused by the excipient glycerol after long-term storage and repeated usage. More attention should be paid to the quality changes of excipients during repeated usage and storage of excipients for the practical purpose. Moreover, we have discovered a novel degradation pathway for insulin and peptides in general. In addition, LC-MS/MS results suggested that the N-terminus of insulin B-chain was prone to chemical degradation which enlightens that it could be potentially modified to improve the stability of insulin formulations. [ABSTRACT FROM AUTHOR]