The impact of extensively used arsenic-containing herbicides on groundwater beneath golf courses has become a topic of interest. Although currently used organoarsenicals are less toxic, their application into the environment may produce the more toxic inorganic arsenicals. The objective of this work was to understand the behavior of arsenic species in percolate water from monosodium methanearsonate (MSMA) applied golf course greens, as well as to determine the influences of root-zone media for United State Golf Association (USGA) putting green construction on arsenic retention and species conversion. The field test was established at the Fort Lauderdale Research and Education Center (FLREC), University of Florida. Percolate water was collected after MSMA application for speciation and total arsenic analyses. The results showed that the substrate composition significantly influenced arsenic mobility and arsenic species transformation in the percolate water. In comparison to uncoated sands (S) and uncoated sands and peat (S + P), naturally coated sands and peat (NS + P) showed a higher capacity of preventing arsenic from leaching into percolate water, implying that the coatings of sands with clay reduce arsenic leaching. Arsenic species transformation occurred in soil, resulting in co-occurrence of four arsenic species, arsenite (As(III)), arsenate (As(V)), monomethylarsonic acid (MMA), and dimethylarsinic acid (DMA) in percolate water. The results indicated that substrate composition can significantly affect both arsenic retention in soil and arsenic speciation in percolate water. The clay coatings on the soil particles and the addition of peat in the soil changed the arsenic bioavailability, which in turn controlled the microorganism-mediated arsenic transformation. To better explain and understand arsenic transformation and transport after applying MSMA in golf green, a conceptual model was proposed.