An aerobic bacterial strain Alcaligenes F-3-4 was isolated from an acrylic fiber wastewater. This strain was able to degrade 2,6-di-tert-butylphenol. The bacterium growth and 2,6-DTBP degradation was determined. Result showed, the lag period was extended when increasing the concentration of 2,6-DTBP. At 37°C and at log period, the degrading rate for 100.0 mg/L 2,6-DTBP could reach 86% within 12 d. Metabolic pathways of 2,6-DTBP degradation by Alcaligenes F-3-4 was studied. The results showed that the intermediate products are acetic acid, pyruvic acid, isobutyric acid, and so on. Identified metabolites and catabolic enzymes illustrate that the used bacterial strain attacks the benzene skeleton at the 2,3- position, and it also demethylates 2,6-DTBP. Metabolic pathways of degradation of 2,6-DTBP are proposed.