In this study, a lab scale EGSB reactor was operated for 400 days to investigate the influence of temperature-decrease on the microbial characteristic of retained sludge. The EGSB reactor was started-up at 15°C seeding with 20°C-grown granular sludge. The influent COD of synthetic wastewater was set at 0.6–0.8 gCOD/L. The process-temperature was stepwise reduced from 15°C to 5°C during 400 days operation. Decrease of temperature of the reactor from 15°C to 10°C caused the decline of COD removal efficiency. However, continuous operation of the EGSB reactor led the efficient treatment of wastewater (70% of COD removal, 50% of methane recovery) at 10°C. We confirmed that the both acetate-fed and hydrogen-fed methanogenic activities of retained sludge clearly increased under 15 to 20°C. Changes of microbial profiles of methanogenic bacteria were analyzed by 16S rDNA-targeted DGGE analysis and cloning. It shows that genus Methanospirillum as hydrogen-utilizing methanogen proliferated due to low temperature operation of the reactor. On the other hand, genus Methanosaeta presented in abundance as acetoclastic-methanogen throughout the experiment.