Aflatoxins cause liver damage and suppress immunity. Through adsorption, smectites can be used to reduce the bioavailability of aflatoxins. To further reduce the toxicity of aflatoxins and to eliminate the treatments of aflatoxin-loaded smectites, the ability to degrade the aflatoxin adsorbed to non-toxic or less toxic compounds is desirable. The objective of the present study was to investigate the effects of temperature and the exchange cation on the transformation of adsorbed aflatoxin B1 on smectite. An AfB1-Ca-smectite (sm) complex was synthesized. To enhance the Lewis acidity of the complexes, the exchanged calcium in the complex was replaced with Mn and Cu to obtain AfB1-Mn-sm and AfB1-Cu-sm complexes, respectively. The aflatoxin-sm complexes and pure aflatoxin B1 were dried at 60°C in aluminum cups, and heated from 100 to 200°C in 25°C steps. Aflatoxin B1 and its transformation products were extracted with methanol after the heat treatment. The extracts were analyzed using UV spectroscopy, high performance liquid chromatography (HPLC)-fluorescence/UV, ultra-performance liquid chromatography (UPLC)-photodiode array (PDA), and electrospray ionization-tandem quadrupole-mass spectrometry (ESI-TQD-MS). The solid residues were analyzed using Fourier-transform infrared spectroscopy (FTIR). The UV and FTIR spectra of the AfB1-sm clay residue extracts obtained after heating had decreased AfB1 peak intensities and shifted peak positions with increased heating temperature. Significant shifts in band positions and changes in the shape of the UV spectra were observed in the extracts from the AfB1-Ca-sm complex heated at 175°C, the AfB1-Cu-sm complex heated at 150°C, and the AfB1-Mn-sm complex heated at 125°C. The HPLC and UPLCMS analyses of AfB1-sm complex extracts indicated a gradual decrease in AfB1 concentration with increased heating temperature and the formation of aflatoxins B2, B2a, M1, M2, and other unidentified compounds. No new compound was observed in the extracts of pure aflatoxin B1 after a comparable heating experiment. These results suggest that smectite can effectively convert aflatoxin to other less toxic forms at elevated temperatures. Smectite ion exchange with Cu or Mn transition-metal cations and heat treatment induced more efficient conversion of the adsorbed aflatoxin B1 molecules to other compounds.