The overuse of glyphosate has resulted in serious environmental contamination. Thus, effective techniques to remove glyphosate from the environment are required. Herein, we isolated a novel strain Stenotrophomonas acidaminiphila Y4B, which completely degraded glyphosate and its major metabolite aminomethylphosphonic acid (AMPA). Y4B degraded glyphosate over a broad concentration range (50−800 mg L −1 ), with a degradation efficiency of over 98% within 72 h (50 mg L −1 ). Y4B degraded glyphosate via the AMPA pathway by cleaving the C−N bond, followed by degradation of AMPA and subsequent metabolism. Y4B demonstrated strong competitiveness and substantially accelerated the degradation of glyphosate in different soils, degrading 71.93 and 89.81% of glyphosate (400 mg kg −1 ) within 5 days in sterile and nonsterile soils, respectively. The immobilized cells of Y4B were more efficient than their free cells and they displayed excellent biodegradation efficiency in a sediment−water system. Taken together, Y4B is an ideal degrader for the bioremediation of glyphosate-contaminated sites.