P-glycoprotein (P-gp) inhibition conduces to improving the ability of chemicals to cross through blood-brain barrier (BBB). The hook-bearing branch of Uncaria is used as a traditional herbal medicine for the treatment of hypertension, headache, stroke, and Alzheimer’s disease in recent years. In this study, the inhibitory effects of 8 alkaloids sourced from Uncaria plants on P-gp were evaluated. Meanwhile, the content of 8 alkaloids in 8 Chinese Uncaria species was quantified simultaneously by high-performance liquid chromatography analysis. Western blotting, real-time quantitative reverse transcription-polymerase chain reaction, and flow cytometry were used to evaluate the P-gp levels in MCF-7/adriamycin-resistant (ADR) cells. It was shown that hirsuteine and hirsutine, selected from 8 candidate alkaloids, could remarkably suppressed P-gp level and function in MCF-7/ADR cells when using Verapamil as positive control. By tracking their chemical distribution in 8 Chinese Uncaria species, we found that hirsuteine and hirsutine were much more abundant in Uncaria rhynchophylla and Uncaria hirsuta than in any other 6 species. And a chemical content profile of these alkaloids was plotted within 8 Chinese species.