Abstract This study attempted to isolate and identify the denitrifying bacteria that utilize acrylonitrile as a substrate from acrylonitrile–butadiene–styrene (ABS) resin wastewater. The performance of the denitrifying bacteria for treating different initial acrylonitrile concentrations was also investigated under anoxic conditions. The results showed that seven strains of denitrifying bacteria that can use acrylonitrile or acrylic acid as a substrate were isolated from the denitrification tank of a wastewater treatment plant in a ABS resin manufacturing plant and a lab-scale anoxic granular activated carbon-fluidized bed. The bacteria strains Acidovorax facilis B and Pseudomonas nautica could utilize acrylonitrile up to 279 mg/l as a substrate for denitrification. For complete nitrate removal, an adequate supply of acrylonitrile was necessary. Under the assumption that the acrylic acid would be completely removed, the removal of 1 mg/l nitrate by A. facilis B or P. nautica, about 0.64–0.74 mg/l acrylonitrile or 0.87–1 mg/l acrylic acid was needed. Because strains A. facilis B and P. nautica could utilize acrylonitrile for denitrification, they are expected to play an important role in the treatment of acrylonitrile in the wastewater treatment plant (denitrification and nitrification processes) and lab-scale granular activated carbon-fluidized bed.