In Multiple Sclerosis (MS), inflammatory demyelinating lesions are associated with a compromised blood-brain barrier (BBB) and with perivascular infiltrations of immune cells into the central nervous system (CNS). Nerve injury-induced protein (Ninjurin)-1 was described as an adhesion molecule that promotes nerve regeneration, in a homotypic Ninjurin-Ninjurin fashion. We recently identified expression of Ninjurin-1 by human brain-derived endothelial cells (ECs) in a proteomic screen for BBB-associated proteins. The goal of this study is to elucidate the role of Ninjurin-1 in the trans-BBB migration of immune cells. We confirmed the expression of Ninjurin-1 on BBB-ECs in vitro by WB and by flow cytometry and we show regulation of Ninjurin-1 upon treatment with pro-inflammatory cytokines TNF and IFN-gamma. Furthermore, Ninjurin-1 immunoreactivity was detected in vitro on BBB-ECs and in situ in MS lesions. Ninjurin-1 was found to be present on human peripheral blood immune cells, and preferentially expressed on CD14 monocytes and CD83 dendritic cells (DCs), as compared to CD4, CD8 or CD19 lymphocytes. We further detected Ninjurin-1 expression on antigen presenting cells, such as CD11c DCs and F4/80 macrophages within the CNS of MOG-induced EAE-affected C57BL/6 mice. Finally, using a modified Boyden chamber assay, we showed that migration of ex vivo CD14 monocytes across the human endothelium is significantly reduced following treatment of BBB-ECs with a competing Ninjurin-1 blocking peptide. Our findings suggest that Ninjurin-1 is a novel adhesion molecule of the CNS endothelium that acts as a regulator of monocyte migration into the brain.