© 2019 The Authors To reveal the metabolic characteristic of organophosphate esters (OPEs) in plants, uptake efficiency, subcellular distribution and hydrolysis of OPEs and their hydrolysis metabolites in wheat (Triticum aestivum L.) were investigated by hydroponic experiments. Tris(2-chloroethyl) phosphate (TCEP) and bis(2-chloroethyl) phosphate (BCEP) were prone to transporting to shoots by transpiration stream, with the translocation factors of 6.2 and 21 for TCEP and BCEP, respectively, as greater than 40% of TCEP and BECP were distributed in the cell sap due to their hydrophilicity. Hydrophobic OPEs (i.e. tri-n-butyl phosphate [TnBP] and triphenyl phosphate [TPhP]), and their hydrolysis metabolites (di-n-butyl phosphate [DnBP] and diphenyl phosphate [DPhP]) were stored in roots, resulting in low translocation capacity in wheat. As organophosphate diesters with relatively high proportions in cell walls (70–84% of DnBP and 41–43% of DPhP) are difficult to being transmembrane transported due to electrical repulsion of the cytomembrane, it is supposed that cell walls could be a main location for in vivo hydrolysis of OPEs. For DnBP, absorption by roots after in vitro hydrolysis of TnBP in hydroponic media was also an important source in wheat. Inhibition experiments showed that it is usually a non-energy-consuming process for root uptake of OPEs and their hydrolysis metabolites. The uptake of OPEs (i.e. TCEP, TnBP, and TPhP) and DPhP were facilitated diffusion mediated by aquaporins or anion channels, while uptake of BCEP and DnBP were simple diffusion processes. This study illustrated the distribution characteristics and translocation capacity of OPEs and their diester metabolites at the subcellular level.