Aspartame is one of the main varieties of artificial sweeteners. Although it has been approved as a food additive, the environmental hazards and ecological risks posed by aspartame are attracting more and more attention. In the present study, strain Pseudarthrobacter sp. AS-1 was isolated and characterized as an efficient aspartame degrader. Strain AS-1 was capable of degrading 200 mg L aspartame within 10 h under conditions optimized at 30 °C and pH 8.0. At the same time, it was found that enzymes degrading aspartame in strain AS-1 were induced and secreted extracellularly. Degradation of aspartame in Pseudarthrobacter sp. AS-1 was identified as following: it was first demethylated to aspartyl-phenylalanine, then degraded to phenylalanine and aspartate, and finally the two amino acids were further degraded. In addition, strain AS-1 was able to remove more than 85% of aspartame in soil and river water. It is the first time that pure bacterial cultures were reported to have the capability of aspartame degradation. These findings add to our knowledge of the microbial metabolic mechanisms of aspartame.