4-(3,4-Dihydroxybenzoyloxymethyl)phenyl- O-beta-D-glucopyranoside (OV-16) is a polyphenolic glycoside isolated from oregano (Origanum vulgare L.), which is a popular Chinese herb and a common spice in Western diet. To understand the biotransformation and pharmacokinetics of OV-16, rats were orally administered OV-16 and oregano decoction. Blood samples were withdrawn at specific time points. The presence of OV-16 and its metabolites protocatechuic acid (PCA) and p-hydroxybenzyl alcohol (HBA) in serum were determined by HPLC method, whereas their conjugated metabolites were assayed indirectly through hydrolysis with beta-glucuronidase and sulfatase. Our results showed that when OV-16 was orally administered, free forms of OV-16, PCA, and HBA were not present in blood and the major metabolites were the glucuronides/sulfates of PCA and HBA sulfate. The serum metabolites of OV-16 exhibited free radical scavenging activity. When oregano decoction was given, the glucuronides and sulfates of PCA were the major metabolites in blood.