A mid-infrared free-electron laser (FEL) is operated as a pulsed and linearly polarized laser with tunable wavelengths within infrared region. Although the FEL can ablate soft tissues with minimum collateral damage in surgery, the potential of FEL for dissecting protein aggregates is not fully understood. Protein aggregates such as amyloid fibrils are in some cases involved in serious diseases. In our previous study, we showed that amyloid-like lysozyme fibrils could be disaggregated into the native form with FEL irradiation specifically tuned to the amide I band (1,620 cm(-1)). Here, we show further evidence for the FEL-mediated disaggregation of amyloid-like fibrils using insulin fibrils. Insulin fibrils were prepared in acidic solution and irradiated by the FEL, which was tuned to either 1,620 or 2,000 cm(-1) prior to the experiment. The Fourier transform infrared spectroscopy (FT-IR) spectrum after irradiation with the FEL at 1,620 cm(-1) indicated that the broad peak (1,630-1,660 cm(-1)) became almost a single peak (1,652 cm(-1)), and the β-sheet content was reduced to 25 from 40% in the fibrils, while that following the irradiation at 2,000 cm(-1) remained at 38%. The Congo Red assay as well as transmission electron microscopy observation confirmed that the number of fibrils was reduced by FEL irradiation at the amide I band. Size-exclusion chromatography analysis indicated that the disaggregated form of fibrils was the monomeric form. These results confirm that FEL irradiation at the amide I band can dissect amyloid-like protein fibrils into the monomeric form in vitro.