The poor bioavailability of mangiferin (MGF) is a major obstacle on its further development. Aimed to illustrate the underlying mechanism and improve its poor exposure, the compared PK profiles of MGF and norathyriol (NTR) after different MGF preparation were performed: pure MGF, the Rhizoma Anemarrhenae (Zhi-mu) decoction, MGF, and timosaponin B2 (TB-2) combination. Furthermore, the potential contributing factors, including uridine diphosphoglucuronosyltransferase (UGT), cytochrome P450 (CYP450), P-gp, and enterobacterial were investigated by comparing the PK profiles with and without the corresponding inhibitors or in different rat models. After taking MGF, CYP450 and UGT inhibition could decrease MGF and NTR exposure; P-gp inhibition slightly enhanced (48%) MGF exposure, whereas more apparent for the improved NTR exposure (302%); enterobacterial inhibition almost completely stopped the NTR production, but no such effect was observed for MGF. Compared with the limited improvement by the abovementioned inhibition, the MGF and NTR exposure could significantly increase by 11.5- and 5.9-fold in the Zhi-mu decoction compared with the MGF treatment, probably contributed to TB-2 as an absorption enhancer because the MGF and TB-2 combination produced a similar level of improvement on the PK paremeters of MGF and NTR to the herb treatment. Likewise, most of the effects by UGT, CYP450, P-gp, and enterobacteria followed a similar variation tendency between them. Therefore, the poor bioavailability of MGF possibly mainly attributed to its poor membrane permeability, but not transporters or metabolic enzymes, and the compatibility of MGF and TB-2 could probably expand the prospective application of MGF by improving its bioavailability. © 2016 BioFactors, 42(5):545-555, 2016.