The effect of aflatoxin B-1 (AFB(1)) degradation by water-assisted microwave irradiation (WMI) and the identification of the degradation products of AFB(1) are key issues in the feasibility study of WMI detoxification. In this study, WMI was applied to degrade pure AFB(1) in aqueous medium, and the kinetics, products, and pathways of the degradation of AFB(1) were studied. The results showed that AFB(1) could be effectively degraded by WMI, and the degradation process followed pseudo-first-order reaction kinetics. In addition, six new main degradation products were identified by ultra-high-performance liquid chromatography Q-Orbitrap mass spectrometry, and a possible degradation pathway was proposed. Furthermore, the variety and quantity of AFB(1) degradation products and the degradation pathway of AFB(1) were affected by microwave heating temperature, time, and power. After WMI treatment, the double bond in the terminal furan ring or the lactone bond of AFB(1) was destroyed, which implied that the toxicity of the degradation product was significantly reduced. Therefore, WMI is deemed to be an effective method to degrade AFB(1).