More than 30 amyloid proteins are reported to be associated with amyloidosis diseases. Studies have implicated histidine may be critically involved in amyloid formation. Here, we used diethylpyrocarbonate (DEPC) modification to obtain a HisB5 mono-ethyloxyformylated insulin (DMI-B5). The secondary structure, amyloidogenicity, metal ion interaction, and cytotoxicity of DMI-B5 and insulin were compared. DMI-B5 was less prone to aggregation in acidic condition but easier to aggregate at neutral pH. DEPC modification resulted in attenuated inhibitory effect of Zn2+ on aggregation, whereas DMI-B5 fibrils induced more severe erythrocytes haemolysis compared to insulin fibrils. This study not only provides a fast new approach for studying the impact of imidazole ring in amyloid formation, but also reveals the critical modulating role of histidine imidazole ring on the amyloidogenicity of insulin.