Background: Oxymatrine is known as one of the most promising alkaloids from Sophora flavescens for its excellent pharmacological effects. Objective: The aim of this research is to assess the biopharmaceutical and pharmacokinetic activities of oxymatrine and clarify its mechanisms of absorption and metabolism. Methods: The biological characteristics of oxymatrine were systematically investigated by UHPLC-MS/MS. The mechanisms of absorption and metabolism of oxymatrine were further clarified through incubation in rat liver microsomes and transport across the Caco-2 monolayer cell absorption model. Results: It was found that the absolute oral bioavailability of oxymatrine was 26.43%, and the pharmacokinetic parameters C-max, T-max, and t(1/2) were 605.5 ng/mL, 0.75 h, and 4.181 h after oral administration, indicating that oxymatrine can be absorbed quickly. The tissue distribution tests showed that oxymatrine distributed throughout all the organs, with the small intestine accumulating the highest level, followed by the kidney, stomach, and spleen. The P-app in Caco-2 cell line absorption model was over 1 x 10(-5) and PDR 1.064, and t(1/2) of oxymatrine in rat liver microsome in vitro was 1.042 h, indicating that oxymatrine can be absorbed easily through passive diffusion and CYP450 enzymes could be involved in its metabolism. The plasma protein binding rate of oxymatrine was 2.78 +/- 0.85%. Conclusion: Oxymatrine can be absorbed into blood easily through passive diffusion, mainly distributed in the intestine, stomach, liver, and spleen in vivo, and CYP450 enzymes in the liver could be involved in its metabolism.