Polyfluoroalkyl phosphate esters (PAPs) have been used in many commercial and industrial applications due to their grease and water repellency, and surfactant properties. However, these compounds yield transformation products such as fluorotelomer alcohols (FTOHs) and perfluorinated carboxylic acids (PFCAs) that are environmentally persistent, bioaccumulative, and potentially toxic. Given that PAPs metabolize to bioactive products, it is important to understand the sites and kinetics of this metabolism. This research compares the biotransformation of a representative PAP, the 8:2 monosubstituted polyfluoroalkyl phosphate (8:2 monoPAP) in typical host biotransformation sites, the liver and intestine, to the mammalian microbiome. The 8:2 monoPAP was incubated in human and rat (male Sprague-Dawley) liver and intestine S9 fractions, and its immediate hydrolysis products, 8:2 fluorotelomer alcohol (8:2 FTOH) was monitored by GC-MS. Human and rat fecal samples were also collected, used as a surrogate for the gastrointestinal microbiome. Enzyme hydrolysis kinetics were measured and compared. Results show that the rat and human gut phosphatases have 2-fold and 1.3-fold more affinity for 8:2 monoPAP transformation, respectively (KM(rat)= (1.2 ± 0.3) ×103nM; KM(human)= 34(1.6 ± 0.4) ×103 nM) compared to liver (KM(rat)= (4.0 ± 1.5) ×103nM; KM(human)= (4.9 ± 3.3) ×103 nM). Results also show the microbiome contributes to 8:2 monoPAP hydrolysis. While the liver and intestine are the primary sites for metabolism, the microbiome playsa role and should not be overlooked. This may impact the relative risk of PAP exposure, given that levels of bioactive products, including PFCAs, may fluctuate depending on environmental and genetic factors leading to microbial diversity across individuals.