For pharmacological treatments of brain disorders, the blood-brain barrier (BBB) poses a formidable obstacle, tightly restricting cerebral uptake of therapeutic agents. Tight junction (TJ) binders, including peptides, proteins and monoclonal antibodies, that specifically bind to the extracellular loops (ECLs) of TJ proteins are increasingly being explored as BBB-weakening agents for enhanced drug delivery to brain. Given that nanobodies (Nbs) can access epitopes that are often inaccessible to conventional antibodies, we immunized alpaca with the ECL of both human (h) and mouse JAM-1 (mJAM1) and obtained 15 JAM1-specific Nbs. These were validated by immunostaining of JAM1-overexpressing cells and mouse brain tissue, and their binding affinity was determined by surface plasmon resonance (SPR) and isothermal titration calorimetry (ITC). A subset was found to weaken the barrier of an eGFP-hCldn5-MDCKII monolayer, as shown by reduced transendothelial electrical resistance (TEER). X-ray crystallography revealed that the lead barrier-weakening Nb, mE6, bound to the membrane-proximal non-dimeric D2 domain of mJAM1. Mutating key residues at this interface eliminated the BBB-weakening effect of mE6, as evidenced by unaltered TEER values and sodium fluorescence (NaFI) leakage in cellular monolayers, compared with nontreated control. This suggests that mE6 does not directly disrupt pre-existing mJAM1 homodimers located in the membrane-distal domain (D1), but instead interferes with the formation of new dimers through steric hindrance, thereby impairing TJ formation and enhancing drug permeability. Our study highlights the potential of alpaca-derived JAM1-targeting Nbs as potential BBB modulators, offering a novel approach for generating TJ-binders to enhance brain drug delivery.