Glyphosate, the most commonly used herbicide in the world, can be degraded into more toxic and persistent products such as aminomethylphosphonic acid (AMPA) or non-toxic products such as sarcosine and glycine. In this study, we used liquid chromatography mass spectrometry (LC-MS) and electrospray ionization (ESI) source Q Extractive Orbitrap mass spectrometry (ESI-Orbitrap MS) to identify glyphosate degradation products and combined with sequential extraction and stable isotopes to investigate the degradation of glyphosate and transformation of phosphorous (P) product in a soil-water system. The LC-MS and ESI-Orbitrap MS results showed that glycine formed during the early stage but was rapidly utilized by soil microorganisms. AMPA started to accumulate at the late stage and was found to be 3–6 times more resistant than glyphosate against degradation; while no sarcosine was formed. The 18O labeling and phosphate oxygen isotope results allowed a clear distinction of the fraction of inorganic P (Pi) derived from glyphosate, about half of which was then rapidly taken up and recycled by soil microorganisms. Our results provide the first evidence of the preferential utilization of glyphosate-derived Pi by microorganisms in the soil-water system. The rapid cycling of Pi derived from this disregarded source has important implications on nutrient management as well as water quality.