A Tsukamurella sp. J8025 with the capability of removing the synthetic dyes (methyl orange) was isolated from activated sludge. The strain J8025 grew well in aerobic and semi-aerobic condition, but the best decolorization was in semi-aerobic culture (static condition). For the optimal decolorization, the most suitable pH and temperature were pH 6.0 and 30°C. More than 80% of methyl orange was decolorized in the LB medium within 4 days of incubation. The decolorization rate could be strongly promoted to over 90% in LB media with glucose within 2 days of incubation. This probability of reason for the consumed glucose as the best energy source was to enhance the cell growth and decolorization activity.