Abstract: To solve the problems existed in breast cancer resistance protein (BCRP), causing the intestinal drug efflux function abnormal under microgravity and the potential impact on therapeutic efficacy and toxicity of substrate drugs, the regulatory mechanism of BCRP function was investigated systematically with a simulated microgravity (SMG) rat ileal tissue model and Caco-2 cell monolayer model, combining with analysis of VEGFR2-PI3K-Akt-NF-κB signaling pathway activity with Western blot and quantitative PCR (qPCR). The key findings include: activating the VEGFR2-PI3K-Akt-NF-κB cascade, SMG can up regulate BCRP protein expression and mRNA levels significantly. The results of functional validation show that intestinal absorption rate can be reduced and intracellular accumulation of substrate drugs can be decreased in SMG groups. The achievement presents a suggestion for oral administration of BCRP substrate drugs under weightlessness environment, that should adjust the dosing regimens distinct from those used under terrestrial conditions. Furthermore, this research can provide hopefully a fundamental data for evaluating pharmacokinetics and toxicity profiles of medications during spaceflight missions, potentially informing medication safety protocols for astronauts in orbit.