Aortic stenosis (AS) is associated with hypofibrinolysis, but its mechanism is poorly understood. We investigated whether LDL cholesterol affects plasminogen activator inhibitor 1 (PAI-1) expression, which may contribute to hypofibrinolysis in AS. Stenotic valves were obtained from 75 severe AS patients during valve replacement to assess lipids accumulation, together with PAI-1 and nuclear factor-κB (NF-κB) expression. Five control valves from autopsy healthy individuals served as controls. The expression of PAI-1 in valve interstitial cells (VICs) after LDL stimulation was assessed at protein and mRNA levels. PAI-1 activity inhibitor (TM5275) and NF-κB inhibitor (BAY 11-7082) were used to suppress PAI-1 activity or NF-κB pathway. Clot lysis time (CLT) was performed to assess fibrinolytic capacity in VICs cultures. Solely AS valves showed PAI-1 expression, the amount of which was correlated with lipid accumulation and AS severity and co-expressed with NF-κB. In vitro VICs showed abundant PAI-1 expression. LDL stimulation increased PAI-1 levels in VICs supernatants and prolonged CLT. PAI-1 activity inhibition shortened CLT, while NF-κB inhibition decreased PAI-1 and SERPINE1 expression in VICs, its level in supernatants and shortened CLT. In severe AS, valvular PAI-1 overexpression driven by lipids accumulation contributes to hypofibrinolysis and AS severity.