Thunbergia laurifolia (TL) has been traditionally used as an antidote and an antipyretic drug by folk healers for centuries in Thailand. Rosmarinic acid (RA) is major compound in TL extract and has attracted great interest due to its potential broad pharmacological effects. Herein, the permeability of RA was investigated in TL extract and as a pure compound in a Caco-2 cell model by using high-performance liquid chromatography with a photodiode array detector (HPLC-PDA). The results reveal that the apparent permeability coefficient (P_app) values of RA in TL extracts and pure RA significantly increased after deconjugation by β-glucuronidase/sulfatase enzymes. Our findings exhibit possible saturable biotransformation of RA and/or membrane transport while penetrated through Caco-2 cells. The cumulative amounts of RA as pure compounds and in TL extracts increased with the exposure time, and the efflux ratio (ER) was 0.27–1.14. RA in the TL extract has a similar absorption in the conjugated form and in the pure compound. The intestinal absorption of them is through passive diffusion. Therefore, our findings conclude that the intestinal transport of RA in TL extracts was mainly penetrated as conjugated forms with glucuronic acid and/or sulfate across Caco-2 cells and transported via passive diffusion.