The endocannabinoid system's significance in maintaining blood-brain barrier (BBB) integrity under physiological and pathological conditions is suggested by several reports, but the underlying molecular mechanisms are not well understood. In this paper, we investigated the effects of depletion of 2-arachidonoylglycerol (2-AG), one of the main endocannabinoids in the central nervous system, on BBB integrity using pharmacological tools. Female Sprague-Dawley rats were injected with the diacylglycerol lipase α (DAGLα) inhibitor LEI-106 (40 mg/kg, i.p.), followed by assessment of BBB integrity via in situ brain perfusion. Liquid chromatography-mass spectrometry, western immunoblotting, light transmittance experiments and pressure myography were also used to further examine the results of DAGLα blockade on the BBB and vascular reactivity. We found that DAGLα inhibition caused BBB opening in cortical brain areas, manifesting as increased sucrose transport measured by in situ brain perfusion. This was accompanied by reduced levels of 2-AG and decreased detection of the tight junction protein zonula occludens-1 (ZO-1). The protein level in cortical areas of neuronal PAS domain protein 4 (NPAS4), encoded by an activity-dependent immediate early gene, was increased without the presence of cortical spreading depression after LEI-106 administration. We also observed a significant increase in pressure-induced constriction within the parenchymal microcirculation after inhibition of DAGLα, possibly altering shear stress in the microcirculation. These results support the role of endogenous 2-AG in maintaining normal tight junction function. This improved understanding of the molecular mechanisms of endocannabinoid system function at the neurovascular unit can help to unlock the therapeutic potentials of cannabinoids in central nervous system disorders associated with BBB dysfunction. KEY POINTS: The administration of the diacylglycerol lipase α (DAGLα) inhibitor LEI-106 (40 mg/kg, i.p.) induced blood-brain barrier (BBB) opening of cortical brain areas in female Sprague-Dawley rats. This BBB disruption was accompanied by reduced levels of 2-arachidonoylglycerol (2-AG) and decreased detection of the tight junction protein zonula occludens-1 (ZO-1). The protein level in cortical areas of neuronal PAS domain protein 4 (NPAS4), encoded by an activity-dependent immediate early gene, was increased without the presence of cortical spreading depression after LEI-106 administration. A significant increase in pressure-induced constriction within the parenchymal microcirculation was also observed after inhibition of DAGLα, possibly altering shear stress. These results support the role of endogenous 2-AG in maintaining normal tight junction function.