Although blood-brain barrier (BBB) integrity is maintained by the cross-talk of endothelial cells, junction proteins, and neurogliovascular network, the epigenetic mechanisms behind BBB permeability are largely unknown. We are reporting for the first time miR29b-mediated regulation of BBB, which is a novel mechanism underlying BBB integrity. We hypothesize that miR29b regulates BBB dysfunction by regulating DNMT3b, which consequently regulates the levels of metalloproteinases, that can eat up the membrane and junction proteins leading to leaky vasculature. In addition, 5'-azacytidine (5'-aza) was used to test its efficacy on BBB permeability. Blood-brain barrier disruption model was created by using homocysteine, and in the models miR29b was identified to be most affected, by using microRNA RT(2)-qPCR array. MiR29b mimics and inhibitors also confirmed that miR29b regulates the levels DNMT3b and MMP9. In hyperhomocysteinemic cystathionine-β-synthase deficient (CBS(+/-)) mice with high brain vessel permeability, miR29b levels were also high as compared with wild-type (WT) mice. Interestingly, 5'-aza improved BBB permeability by decreasing the expression of miR29b. In conclusion, our data suggested miR29b-mediated regulation of BBB dysfunction through DNMT3b and MMP9. It also potentiates the use of microRNAs as candidates for future epigenetic therapies in the improvement of BBB integrity.