To investigate the effect of RMP-7 and its derivative on drug transport across blood brain barrier (BBB), RMP-7 and DSPE-PEG-NHS [1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-n-[poly(ethyleneglycol)]-hydroxy succinamide, PEG M 3400] were conjugated under mild conditions and the reaction ratio was determined using MALDI-TOF-MS (matrix-assisted laser desorption-ionization time-of-flight mass spectrometry). An endothelial cell monolayer in vitro BBB model was established and used to determine the bioactivity of RMP-7 and its derivative "opening BBB" Horse radish peroxide (HRP), liposome (HRP-L-PEG), and Evens blue (EB) liposome (EB-L-PEG) were prepared using the reverse-phase evaporation method. HRP-L-PEG-RMP-7 and EB-L-PEG-RMP-7 were obtained by inserting DSPE-PEG-RMP-7 into the surface of liposome. The bioactivity of RMP-7 and DSPE-PEG-RMP-7 opening BBB were evaluated to determine their effect on the permeation ratio of HRP and HRP liposome across the in vitro BBB model. To evaluate the in vivo bioactivity of RMP-7 and DSPE-PEG-RMP-7 on EB transport across BBB into the brain, the indicated compounds were administered to rats. Then, brain slices were analyzed using confocal laser scanning microcopy and the EB concentration in the brain, liver, spleen, lung, and kidney was determined using the formamide-extraction-ultraviolet-spectrophotometric method. The results demonstrated that RMP-7 was conjugated with DSPE-PEG-NHS at the molecular ratio of 1:1 and the product is DSPE-PEG-RMP-7. Compared with adding HRP alone, RMP-7 and DSPE-PEG-RMP-7 improved 2- to 3-fold the transport of HRP in the in vitro BBB model. The in vivo experiments showed that DSPE-PEG-RMP-7 was better at facilitating EB transport into brain than RMP-7. The reason may be that DSPE-PEG-RMP-7 can "open BBB" as soon as the EB-L-PEG-RMP-7 reaches BBB.