(1) Background: Traumatic brain injury (TBI) frequently occurs worldwide, resulting in high morbidity and mortality. Here, we hypothesized that TBI impairs an autoregulatory mechanism, namely the flow-induced constriction of isolated rat middle cerebral arteries (MCAs). (2) Methods: TBI was induced in anaesthetized rats by weight drop model, and then MCAs were isolated and transferred into a pressure-flow chamber. The internal diameter was measured by a video-microscopy. (3) Results: In MCAs from intact rats, increases in flow and pressure + flow elicited constrictions (−26 ± 1.9 µm and −52 ± 2.8 µm, p < 0.05), which were significantly reduced after TBI or in the presence of thromboxane-prostanoid (TP receptor) antagonist SQ 29,548. Flow-induced constrictions were significantly reduced by HET0016, inhibitor of cytochrome P450 4A (CYP450 4A). Arachidonic acid, (AA, 10⁻⁷ M), and CYP-450 4A metabolite 20-hydroxyeicosatetraenoic acid (20-HETE) elicited constrictions of intact MCA (−26 ± 2.3% and −31 ± 3.6%), which were significantly reduced after TBI (to 11 ± 1.3% and −16 ±2.5%). The TP receptor agonist U46619 (10⁻⁷ M) elicited substantial constrictions of MCA from intact rats (−21 ± 3.3%), which were also significantly reduced, after TBI (to −16 ± 2.4%). (4) Conclusions: Flow-induced constrictor response of MCA is impaired by traumatic brain injury, likely due to the reduced ability of cytochrome P450 4A to convert arachidonic acid to constrictor prostaglandins and the mitigated sensitivity of thromboxane-prostanoid receptors.