Sepsis-associated encephalopathy (SAE) is a diffuse dysfunction of the central nervous system and a major manifestation of sepsis, associated with acute clinical deterioration and poor prognosis. Despite advances in understanding its pathophysiology and sepsis-induced cerebral alterations, effective pharmacological treatments remain unavailable. Cyclin-dependent kinase 9 (CDK9) is a ubiquitous kinase implicated in several inflammatory processes, however, its role in sepsis remains poorly understood. This study investigated whether CDK9 activity contributes to SAE pathophysiology and whether its pharmacological inhibition confers protection in a clinically relevant sepsis model. Polymicrobial sepsis was induced in male C57BL/6OlaHsd mice using caecal ligation and puncture (CLP). One hour after the procedure, mice received the selective CDK9 inhibitor LDC000067 (50 mg/kg i.v.) or vehicle. At 24 h, sepsis induced marked CDK9 activation in the prefrontal cortex, accompanied by neuroinflammation, evidenced by microglial activation, and blood-brain barrier (BBB) disruption, reflected by increased plasma neurofilament light chain levels. Treatment with LDC000067 attenuated septic alterations both centrally and peripherally, reducing multiorgan dysfunction, the systemic cytokine storm, and clinical severity. In vitro , exposure of human microglial cells to a sepsis-like inflammatory stimulus increased NF-κB-related transcriptional responses, which were significantly reduced by CDK9 inhibition. Similarly, exposure of brain microvascular endothelial cells to the same stimulus resulted in endothelial barrier dysfunction, mitigated by CDK9 inhibition through preservation of claudin-5 expression and barrier integrity. Altogether, these findings identify a previously unreported role for CDK9 in SAE pathogenesis and show that its pharmacological inhibition attenuates key features of SAE, including microglial activation and BBB disruption.