Blood-brain barrier (BBB) integrity is one of the important elements of central nervous system (CNS) homeostasis. MicroRNAs (miRs) have been demonstrated to play a role in many CNS disorders such as stroke and traumatic brain injury. MiR-212/132 are highly expressed in the CNS but their role at the BBB has not been characterized yet. Thus, we analyzed the expression of miR-212/132 in hypoxic mouse and human brain microvascular endothelial cells (BMEC) as well as in posttraumatic mouse and human brain tissue and serum exosomes. MiR-212/132 expression was detected in brain capillaries by in situ hybridization and was increased up to ten times in hypoxic BMEC. Over-expression of pre-miR-212/132 in BMEC decreased barrier properties and reduced migration of BMEC in the wound healing assay. We identified and validated tight junction proteins claudin-1 (Cldn1), junctional adhesion molecule 3 (Jam3), and tight junction-associated protein 1 (Tjap1) as potential miR-212/132 targets. Over-expression of miRs led to a decrease in mRNA and protein expression of Cldn1, Jam3, and Tjap1, which could be rescued by a respective anti-miR. In conclusion, our study identifies miR-212/132 as critical players at the hypoxic BBB. In addition, we propose three new direct miR-212/132 targets to be involved in miR-212/132-mediated effects on BBB properties.