After spinal cord injury, the disruption of blood-spinal cord barrier by activation of matrix metalloprotease is a critical event leading to infiltration of blood cells, inflammatory responses and neuronal cell death, contributing to permanent neurological disability. Recent evidence indicates that fluoxetine, an anti-depressant drug, is shown to have neuroprotective effects in ischaemic brain injury, but the precise mechanism underlying its protective effects is largely unknown. Here, we show that fluoxetine prevented blood-spinal cord barrier disruption via inhibition of matrix metalloprotease activation after spinal cord injury. After a moderate contusion injury at the T9 level of spinal cord with an infinite horizon impactor in the mouse, fluoxetine (10 mg/kg) was injected intraperitoneally and further administered once a day for indicated time points. Fluoxetine treatment significantly inhibited messenger RNA expression of matrix metalloprotease 2, 9 and 12 after spinal cord injury. By zymography and fluorimetric enzyme activity assay, fluoxetine also significantly reduced matrix metalloprotease 2 and matrix metalloprotease 9 activities after injury. In addition, fluoxetine inhibited nuclear factor kappa B-dependent matrix metalloprotease 9 expression in bEnd.3, a brain endothelial cell line, after oxygen-glucose deprivation/reoxygenation. Fluoxetine also attenuated the loss of tight junction molecules such as zona occludens 1 and occludin after injury in vivo as well as in bEnd.3 cultures. By immunofluorescence staining, fluoxetine prevented the breakdown of the tight junction integrity in endothelial cells of blood vessel after injury. Furthermore, fluoxetine inhibited the messenger RNA expression of chemokines such as Groα, MIP1α and 1β, and prevented the infiltration of neutrophils and macrophages, and reduced the expression of inflammatory mediators after injury. Finally, fluoxetine attenuated apoptotic cell death and improved locomotor function after injury. Thus, our results indicate that fluoxetine improved functional recovery in part by inhibiting matrix metalloprotease activation and preventing blood-spinal cord barrier disruption after spinal cord injury. Furthermore, our study suggests that fluoxetine may represent a potential therapeutic agent for preserving blood-brain barrier integrity following ischaemic brain injury and spinal cord injury in humans.