We previously reported that trans-10-hydroxy-2-decenoic acid (10-HDA), the exclusive lipid component of royal jelly (RJ), alleviates Lipopolysaccharide (LPS)-induced neuroinflammation both in vivo and in vitro. However, whether 10-HDA can protect against LPS-induced blood-brain barrier (BBB) damage is largely unexplored. In this study, we first observed that 10-HDA decreased BBB permeability in LPS-stimulated C57BL/6 mice by Evan's blue (EB) dye. Immunostaining and Western blot results showed that 10-HDA alleviated BBB dysfunction by inhibiting the degradation of tight junction proteins (occludin, claudin-5 and ZO-1). In LPS-stimulated human brain microvascular endothelial cells (HBMECs), 10-HDA decreased the expression of chemokines (CCL-2 and CCL-3), adhesion molecules (ICAM-1 and VCAM-1), reactive oxygen species, matrix metalloproteinases (MMP-2 and MMP-9) and increased the expression of tight junction proteins. Interestingly, LC-MS/MS analysis showed that 10-HDA pretreatment upregulated the expression of mitochondria-associated proteins, which may reflect the mechanism underlying the regulatory effect of 10-HDA on reactive oxygen species. We further illustrated that 10-HDA promoted the activation of the AMPK pathway and the downstream PI3K/AKT pathway. Compound C (an AMPK inhibitor) and LY294002 (a PI3K inhibitor) markedly reversed the alleviating effect of 10-HDA on the expression of tight junction proteins, indicating that 10-HDA inhibited LPS-induced BBB dysfunction by triggering the activation of the AMPK/PI3K/AKT pathway. Collectively, these data reveal that 10-HDA may be an interesting candidate for clinical evaluation in the treatment of diseases related to BBB damage.