Background and Purpose Blood–brain barrier (BBB) breakdown is one of the crucial pathological changes of cerebral ischaemia–reperfusion (I/R) injury. Trilobatin (TLB), a naturally occurring food additive, exerts neuroprotective effects against cerebral I/R injury as demonstrated in our previous study. This study was designed to investigate the effect of TLB on BBB disruption after cerebral I/R injury. Experimental Approach Rats with focal cerebral ischaemia caused by transient middle cerebral artery occlusion were studied along with brain microvascular endothelial cells and human astrocytes to mimic BBB injury caused by oxygen and glucose deprivation/reoxygenation (OGD/R). Key Results The results showed that TLB effectively maintained BBB integrity and inhibited neuronal loss following cerebral I/R challenge. Furthermore, TLB increased tight junction proteins including ZO‐1, Occludin and Claudin 5, and decreased the levels of apolipoprotein E (APOE) 4, cyclophilin A (CypA) and phosphorylated nuclear factor kappa B (NF‐κB), thereby reducing proinflammatory cytokines. TLB also decreased the Bax/Bcl‐2 ratio and cleaved‐caspase 3 levels along with a reduced number of apoptotic neurons. Molecular docking and transcriptomics predicted MMP9 as a prominent gene evoked by TLB treatment. The protective effects of TLB on cerebral I/R‐induced BBB breakdown was largely abolished by overexpression of MMP9, and the beneficial effects of TLB on OGD/R‐induced loss of BBB integrity in human brain microvascular endothelial cells and astrocyte co‐cultures was markedly reinforced by knockdown of MMP9. Conclusions and Implications Our findings reveal a novel property of TLB: preventing BBB disruption following cerebral I/R via targeting MMP9 and inhibiting APOE4/CypA/NF‐κB axis. ∎