Concentrations of low-density lipoprotein (LDL) above 0.8 mg/ml have been associated with increased risk for cardiovascular diseases and impaired endothelial functionality. Here, we demonstrate that high concentrations of LDL (1 mg/ml) decreased NOS3 protein and RNA levels in primary human endothelial cells. In addition, RNA sequencing data, in particular splice site usage analysis, showed a shift in NOS3 exon-exon junction reads towards those specifically assigned to nonfunctional transcript isoforms further diminishing the functional NOS3 levels. The reduction in NOS3 was accompanied by decreased migratory capacity, which depends on intact mitochondria and ATP formation. In line with these findings, we also observed a reduced ATP content. While mitochondrial mass was unaffected by high LDL, we found an increase in mitochondrial DNA copy number and mitochondrial RNA transcripts but decreased expression of nuclear genes coding for respiratory chain proteins. Therefore, high LDL treatment most likely results in an imbalance between respiratory chain complex proteins encoded in the mitochondria and in the nucleus resulting in impaired respiratory chain function explaining the reduction in ATP content. In conclusion, high LDL treatment leads to a decrease in active NOS3 and dysregulation of mitochondrial transcription, which is entailed by reduced ATP content and migratory capacity and thus, impairment of endothelial cell functionality.