Neolamarckia cadamba (Roxb.) Bosser (Rubiaceae) is a fast-growing and economically important tree in Asia, which has a wide range of biological and pharmacological activities. Using the UPLC-MS fingerprints of the barks of N. cadamba that sampled from 12 different provenance (10 trees per provenance) in a provenance trail, the difference in the components of these samples was visualized. Analyzing the spectrum data and the cytotoxic activity, the relationships of chemical composition and in vitro anti-tumor activity had been established. It was noted that 3α-dihydrocadambine (3a-Dhc), one of the characteristic components in N. cadamba, could bind to the active pocket of P-glycoprotein (P-gp), and then inhibit its efflux activity, leading to reverse the adriamycin (ADR) resistance in MCF7/ADR cells with no toxicity. Taken together, our data pointed out that N. cadamba in different families had high but different contents of cadambine alkaloids, and the UPLC-MS fingerprint could be a powerful tool for guiding the variety breeding, and Sample 12, the provenance from Mengla County, may be a valued source for the best prospect of anti-tumor application. More importantly, 3a-Dhc could be a potential lead for developing a P-gp inhibitor with high potency and low toxicity. © 2022 Elsevier B.V.