Jatrorrhizine is a protoberberine alkaloid derived from Coptis chinensis concentrated extremely in rat kidney. In the present study, the involvement of rat organic cation transporter 2 (rOCT2) in the renal uptake of jatrorrhizine in rat was investigated through in vitro and in vivo experiments. Saturable and nonsaturable uptakes of jatrorrhizine were observed in rat kidney slices and rOCT2-Madin-Darby canine kidney (MDCK) cells. Michaelis-Menten constants of 677.8 and 21.0 µM, maximum uptake rate of 123 (pmol/min)/mg kidney and 13.7 (pmol/min)/mg protein, and nonsaturable uptake clearance of 0.054 (µL/min)/mg kidney and 0.032 (µL/min)/mg protein were observed in rat kidney slices and rOCT2-MDCK cells, respectively. As inhibitors of rOCT2, corticosterone, verapamil, and cimetidine can inhibit jatrorrhizine uptake in rat kidney slices and rOCT2-MDCK cells. Their median inhibitory concentration in rat kidney slices was 7, 78, and 538 µM, whereas that in rOCT2-MDCK cells was 1.07, 86.5, and 151.8 µM. Coadministration with 20 mg/kg corticosterone, a selective inhibitor of rOCT2, reduced the jatrorrhizine concentration in the cortex and medulla in the in vivo experiment. Thus, rOCT2 is mainly responsible for the renal uptake of jatrorrhizine in rat and in the regulation of jatrorrhizine concentration in the kidney.