Soil acid phosphatases (APs), secreted by plant roots and microorganisms, are critical to the recycling of inorganic phosphate (Pi) from organic matter. Accordingly, up-regulation of gene expressions of APs occurs in Pi-deficient soils. Much remains to be elucidated to evaluate this critical role of AP-mediated phosphorous (P) recycling. The important knowledge gaps addressed here include elucidating the reactivity of APs towards mixtures of organic phosphorylated (Porg) compounds, determining substrate specificity of APs from different biological hosts for phosphorylated biopolymers, and unraveling how binding of APs to soil mineral surfaces influences the activity of APs. In Chapter 1, we employed high resolution liquid chromatography-mass spectrometry (LC-MS) to explore the reactivity of a fungal phytase towards a heterogeneous mixture containing different Porg compounds. The internal competition of substrates in the mixture led to lower dephosphorylation rates compared to single substrate reactions, particularly for multi-phosphorylated substrates. Molecular docking simulations revealed that the higher density of negative charges in the substrate caused more favorable interaction dynamics in the active site. In Chapter 2, we combined LC-MS, 31P nuclear magnetic resonance (NMR) spectroscopy, and molecular simulations to investigate the kinetic rates, specificities, and substrate-enzyme interactions in the dephosphorylation of polyphosphate (polyP) and RNA by plant and fungal APs. Fungal AP preferred polyP more than plant purple AP (PAPs), but plant PAP dephosphorylated RNA faster than fungal AP. Molecular modeling showed that the preference of an AP for a specific P-polymer was determined by the number of interactions and the orientation of the target phosphate moiety with respect to the catalytic center. In Chapter 3, we combined atomic force microscopy-dynamic force spectroscopy with Fourier-transform infrared spectroscopy, and 1H NMR to investigate the adsorption mechanism, conformational changes, and reactivity of a fungal phytase upon adsorption to soil minerals: illite, goethite, and quartz. Compared to illite, binding on quartz and goethite surfaces induced greater conformational changes in the enzyme characterized by decrease in α-helix and β-sheet contents in the secondary structure, resulting in greater decrease in the enzyme activity. Collectively, the findings from this research provide a molecular-level understanding of the enzymatic P recycling processes in soil.