To investigate the role of placental growth factor/vascular endothelial growth factor (PlGF-VEGF) hetemdimers are involved in the blood-retinal barrier (BRB) breakdown and the associated mechanism, human retinal endothelial cells (HRECs) were treated with recombinant human (rh)PlGF-VEGF hetemdimers and rhPlGF and studied in normal and high-glucose conditions. HREC barrier function was evaluated by the measurement of trans-endothelial electrical resistance (TEER). Adeno-Associated Virus Type 5 (AAV5) vectors overexpressed PlGF in the retina by intravitreal injection into the C57BL6 mouse eye. AAV5-GFP vector and naive animals were used as controls. Immunofluorescence (IF) and western blots examined the protein expression of PlGF-VEGF hetemdimers, VEGF, PlGF, NF kappa B, p-I kappa B alpha, ZO-1, and VE-cadherin in HREC and mouse retina. PlGF-VEGF heterodimers were detected predominantly in the HREC cell nuclei based on IF and cytoplasmic and nuclear fractionation experiments. High glucose treatment increased PlGF-VEGF nuclear abundance. Dot immunoblotting demonstrated a strong affinity of the 5D11D4 antibody to PlGF-VEGF heterodimers. rhPlGF-VEGF disrupted the barrier function of HREC, which was prevented by the neutralization of PlGF-VEGF by the 5D11D4 antibody. Stimulation of HRECs with rhPlGF also led to an increase in the nuclear signals for PlGF-VEGF, p-I kappa B alpha, and colocalization of NF kappa B p65 and PlGF-VEGF in the nuclei. The selective IKK2 inhibitor IMD0354 disrupted the nuclear colocalization. Treatment with IMD0354 restored the barrier function of HREC, as indicated by the ZO-1 and VE-cadherin expression. In the mouse retinas, P1GF overexpression by AAV5 vector reduced ZO-1 expression and increased abundance of pI kappa B alpha. PIGF/VEGF heterodimers mediate BRB breakdown potentially through the canonical NF kappa B activation.