Marinobufagenin (MBG) is a cardiotonic steroid that increases in the circulation in preeclampsia. Preeclampsia and eclampsia are associated with cerebral edema. Therefore, we examined the effects of MBG on human brain microvascular endothelial cells (HBMEC) in vitro. MBG enhanced the permeability of HBMEC monolayers at 1, 10 and 100 nM doses, but had no effect at 0.1 nM. Agilent Human Gene Expression microarrays were utilized in these studies. MBG treatment (10 nM for 12 h) down-regulated concentrations of the soluble VEGFR transcript sFLT by 59%, but did not alter those of FLTv3 mRNA (determined by quantitative PCR). When treated and control HBMEC transcriptomes were interrogated on microarrays, 1069 genes appeared to be regulated by MBG. Quantitative RT-PCR confirmed that MBG treatment up-regulated ENKUR mRNA concentrations by 57%. Its protein product interacts with calmodulin and calcium channel proteins. MBG treatment down-regulated several genes whose protein products are involved in cell adhesion (ITGA2B, FERMT1, CLDN16 and TMEM207) and cell signaling (GRIN2C, SLC8A1 and ESR1). The level of down-regulation ranged from 22 to 66%. Altogether, MBG actively enhanced the permeability of HBMEC monolayers while down-regulating genes involved in adhesion. MBG treatment had variable effects on ENKUR, GRIN2C and SLC8A1 genes, all associated with calcium transport. These studies provide the basis for future investigations of MBG actions in normal physiology and disease.