Traumatic brain injury (TBI) leads to permanent damage, including neurological deficits, cognitive deficits, and cerebral edema. The specific inhibitor of serine protease Omi/high-temperature requirement A2 (Omi/HtrA2), UCF-101, exerts neuroprotective effects, but its role in TBI remains unclear. Eighty-four male Sprague Dawley rats were randomized to control, TBI, UCF-101 of low dose (1.5 μmol/kg), middle dose (3.0 μmol/kg), and high dose (6.0 μmol/kg), Compound C (AMPK inhibitor, 20 mg/kg), and high dose + Compound C groups. TBI rat modeling was operated by the controlled cortical impact method. Modified neurological severity score (mNSS) cognitive function, cerebral edema index, hematoxylin-eosin staining, TUNEL staining for apoptosis, ethidium bromide staining for blood-brain barrier (BBB) permeability, enzyme-linked immunosorbent assay for inflammation response, and Western blot analysis were performed. In TBI rats, UCF-101 caused decreased mNSS score, brain edema, neuronal apoptosis, as well as P-NF-κBp65/NF-κBp65, tumor necrosis factor-α, interleukin (IL)-1β, and IL-8 expression, while P-AMPK/AMPK, zonula occludens protein, Occludin, and Claudin-5 expression increased, accompanied with up-regulated cognitive function. Moreover, Compound C further exacerbated brain tissue lesions, neuronal damage, inflammation response, and neuronal apoptosis, while high-dose UCF-101 offset its effect. UCF-101 may inhibit apoptosis and BBB permeability to exert neuroprotective effects in TBI rats by regulating the AMPK/NF-κB pathway, advancing UCF-101 clinical applications for TBI treatment.