(±)-Praeruptorin A (dl-PA) is one of the main pyranocoumarins of Peucedani Radix and the chemical marker for quality control of the herb in China. This study investigated the transport and metabolism of dl-PA, for the first time, in Caco-2 cell monolayers. PA enantiomers of dl-PA in the transport study were simultaneously determined using a simple and rapid enantio-selective high performance liquid chromatography-UV method. Both dextrorotatory (dPA) and levorotatory (lPA) enantiomers traversed Caco-2 monolayer rapidly in both directions (absorptive Papp 2.013.03×10-5 cm/s; secretory Papp: 1.581.96×10-5 cm/s) mainly via passive diffusion. Higher transport rates of dPA were observed in both directions. PA enantiomers were incompletely recovered after the transport study. Nonspecific binding to the Transwell inserts, irreversible binding to cellular components and metabolism within the cells accounted for the loss. dl-PA was partially hydrolyzed in Caco-2 monolayers and yielded two stereoisomers via removal of the acetyl group from C-4′ position. Both phenylmethylsulphonyl fluoride (a nonspecific esterase inhibitor) and bis(p-nitrophenyl) phosphate sodium salt (a specific inhibitor of carboxylesterases) completely abolished dl-PA hydrolysis. In summary, PA enantiomers were rapidly transported across Caco-2 cells and partially hydrolyzed by carboxylesterases during permeation. These findings provide mechanistic understanding of in vivo pharmacokinetic properties of dl-PA. © 2011 Informa UK, Ltd.