This study investigated the metabolism of salvianolic acid A (SAA) both in vivo and in vitro. Liquid chromatography-mass spectrometry analysis of drug-containing rat bile samples and bile samples hydrolyzed by glucuronidase revealed a series of methylated conjugates of SAA and its glucuronides, as well as the predominance of the methylation pathway of SAA in rats. For the first time, four major methylated metabolites present in vivo were prepared for structure characterization and bioactivity evaluation using in vitro coincubation systems with rat hepatic cytosol protein as the enzyme donor. By using nuclear magnetic resonance imaging and other spectroscopic methods, these metabolites were unambiguously elucidated as 3-O-methyl-SAA (M1), 3'-O-methyl-SAA (M2), 3,3″-O-dimethyl-SAA (M3), and 3',3″-O-dimethyl-SAA (M4), respectively. Along with results from the enzyme inhibition study, selective formation of these meta-O-methylated derivatives indicated that catechol O-methyltransferase (COMT) is responsible for methylated transformation of SAA. All of these metabolites displayed fairly high antioxidant potency against in vitro rat liver lipid peroxidation with half-maximal inhibitory concentrations that were much lower than those of the positive controls and even SAA. Overall, the results from this study demonstrate that SAA is a metabolically unstable compound that undergoes rapid methylation metabolism catalyzed by COMT, and these generated O-methylated metabolites may be largely responsible for its in vivo pharmacological effects.