Glyphosate is a globally dominant herbicide. Here, we studied the degradation and microbial response to glyphosate application in a wetland soil in central Delaware for controlling an invasive species (Phragmites australis). We applied a two-step solid-phase extraction method using molecularly imprinted polymers designed for the separation and enrichment of glyphosate and aminomethylphosphonic acid (AMPA) from soils before their analysis by ultra-high-performance liquid chromatography (UHPLC) and a Q Exactive Orbitrap mass spectrometry methods. Our results showed that approximately 90% of glyphosate degraded over 100 d after application, with AMPA being a minor (<10%) product. Analysis of glyphosate-specific microbial genes to identify microbial response and function revealed that the expression of the phnJ gene, which codes C-P lyase enzyme, was consistently dominant over the gox gene, which codes glyphosate oxidoreductase enzyme, after glyphosate application. Both gene and concentration data independently suggested that C-P bond cleavage—which forms sarcosine or glycine—was the dominant degradation pathway. This is significant because AMPA, a more toxic product, is reported to be the preferred pathway of glyphosate degradation in other soil and natural environments. The degradation through a safer pathway is encouraging for minimizing the detrimental impacts of glyphosate on the environment. Environmental ImplicationsGlyphosate is a globally dominant herbicide. No viable market replacement has forced the scientific community to search for ways for its safer degradation. This study identified a naturally occurring safer pathway for the degradation of glyphosate in the soil where its most common degradation product, aminomethylphosphonic acid (AMPA), is severely suppressed over the sarcosine/glycine, leading to a safer product pathway of degradation. Identification of a naturally occurring approach that biases degradation towards benign products is highly sought for minimizing detrimental impacts on the environment.