Highlights ► Homocysteine thiolactone (HCTL) augmented rate of insulin unfolding, increased solvent-exposed hydrophobic regions and induced a transition from α-helix into predominantly β-sheet structure. ► HCTL markedly enhanced the quantity of insulin fibril formation in both agitating and non-agitating systems. ► Insulin fibrils obtained in the presence of HCTL and those collected earlier in the pathway of insulin fibrillation displayed higher cytotoxicity against cancer cells. ► Results of this study may suggest HCTL as a possible contributing factor to the pathology of insulin fibrils.