Laboratory bioregeneration tests for brewery spent diatomite with the ammonifying bacteria Lysini-bacillus fusiformis were conducted. After a 14-day incubation, 51% of the proteins accumulated in spent diatomite were degraded. Scanning electron microscopy (SEM), Fourier transform infrared spectrometry (FTIR) and Brunauer-Emmett-Teller (BET) analysis showed that the surface performance and pore structure of spent diatomite after bioregeneration were improved markedly. Adsorption experiments for methylene blue (MB) and Cr(III) of diatomite regenerated via the biological method showed that the removal degrees of MB and Cr(III) reached 95.5% and 71.7%, respectively, which were significantly higher than those obtained with the thermal regeneration method. These results illustrated that the biological method was better able to improve the adsorption ability of spent diatomite. Investigations on adsorption mechanisms confirmed that physical adsorption related to the porous structure of diatomite influenced both MB and Cr(III) adsorption. In addition, the hydroxyl group on the diatomite surface were critical for MB adsorption, and chemical adsorption associated with the ion exchange process might be an important sorption mechanism of Cr(III). (C) 2019 Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.