We investigated the enhancement of biogas production from anaerobic digestion of sewage sludge with the addition of an aerobic thermophilic bacterial culture of Geobacillus sp. strain AT1. Biogas production was significantly enhanced by the 2 ml addition of stationary-growth-phase culture (24h) of strain AT1 to 38 ml methanogenic sludge. Strain AT1 grew aerobically in F-JX synthetic medium, and optimum growth occurred at pH 7.0 and 65°C with shaking at 140 rpm. The optimum temperature for protease activity in the strain AT1 culture was around 65°C in the presence of 2 mM CaCl₂, where relative protease activity reached 210% compared with that at 35°C without CaCl₂. Protease activity was stable from 35 to 65°C, but rapidly decreased within 30 min incubation at 85°C. A protease inhibitor mixture (AEBSE, bestatin, E-64, EDTA, and pepstatin) inhibited the biogas enhancement by the strain AT1 culture. Methane production was enhanced by 2.1 times with the addition of the strain AT1 culture compared with the addition of F-JX medium, and was enhanced by 2.8 times with the additions of authentic protease and serine protease S. These results suggest that the enhancement of biogas production from anaerobic digestion of sewage sludge was caused by the protease activity of aerobic thermophilic Geobacillus sp. strain AT1.