Insulin fibril formation decreases the effectiveness of insulin therapy and causes amyloidosis in diabetes. Studies suggest that phytochemicals are capable of inhibiting fibril formation. Herein, we investigated the inhibitory effects of anthocyanins, including cyanidin, cyanidin-3-glucoside (C3G), cyanidin-3-rutinoside (C3R), malvidin, and malvidin-3-glucoside (M3G) on fibril formation. Our results revealed that anthocyanins (50-200 μM) significantly reduced the formation of insulin fibrils by increasing lag times and decreasing ThT fluorescence at the plateau phase. These findings were confirmed by TEM images, which showed reduced fibril length and number. Furthermore, FTIR analysis indicated that anthocyanins reduced the secondary structure transition of insulin from α-helix to β-sheet. Anthocyanins interacted with monomeric insulin (residues B8-B30) via H-bonds, van der Waals, and hydrophobic interactions, covering the fibril-prone segments of insulin (residues B12-B17). Based on the structure-activity analysis, the presence of glycosides and hydroxyl groups on phenyl rings increased intermolecular interaction, mediating the inhibitory effect of anthocyanins on fibril formation in the order of malvidin < cyanidin < M3G < C3G < C3R. Moreover, anthocyanins formed H-bonds with preformed insulin fibrils, except for malvidin. In preadipocytes, C3R, C3G, and cyanidin attenuated insulin fibril-induced cytotoxicity. In conclusion, anthocyanins are effective inhibitors of insulin fibril formation and cytotoxicity.