The use of glyphosate-resistant crops enabled the adoption of no-till (NT) conservation management cropping systems. This study determined the fate of glyphosate in a Dundee silt loam at soil depths of 0 to 2 cm and 2 to 10 cm from test plots under continuous conventional-tillage (CT) or NT soybean for ten years. Soils were assessed for soil biological properties, fluorescein diacetate (FDA) hydrolysis, and soil microbial community structure based on fatty acid methyl ester (FAME) analysis. Mineralization and extractable ¹⁴C determined in soils treated with ¹⁴C-glyphostae over 35 days. Surface NT soils had the highest organic matter content, FDA hydrolytic activity and unique FAME profiles compared to either depth of CT soil or the lower 2-10 cm of NT soil. The most rapid initial glyphosate mineralization was observed in the surface NT soil and was correlated with the highest soil organic matter content and FDA hydrolysis. After five weeks, 70% of the glyphosate was mineralized in surface NT, 63% in either CT depth compared to 51% in the lower NT soil, while 28% of ¹⁴C was recovered by NaOH extraction in the lower depth of NT soils compared to 20% in CT soils or 15% in NT surface soil. The potential for sorption based on Kd (batch equilibrium) was NT 0 to 2 cm = 58, NT 2 to 10 cm = 42, compared to 19 and 21 for CT in the upper and lower depth. Despite greater glyphosate sorption and lower bioavailability in surface NT soil, glyphosate dissipation was more rapid owing presumably to a more active and diverse microbial community coupled with a greater abundance of available carbonaceous substrates. These data indicate glyphosate runoff or leaching potential is likely reduced in NT systems due to a more rapid dissipation and greater sorption compared to CT systems.