Despite Paraquat (PQ) being banned in several countries, it is still one of the most commonly used herbicides in agriculture. This compound is known to induce damaging effects on human and animal brain cells by generating Reactive Oxygen Species (ROS). However, there is few evidence of PQ effect on Human Brain Microvascular Endothelial Cells (HBMECs), one of the major component of the Blood–Brain Barrier (BBB). The present study aimed at unraveling biological mechanisms associated to the exposure of 1, 10 and 100 µM of PQ for 24 h on HBMECs. High-throughput mass spectrometry-based proteomics using data-independent acquisition (DIA) was applied. Biological pathway enrichment and cellular assays such as mitochondrial respiration and cholesterol level were performed to verify proteomics results. A total of 3753 proteins were quantified out of which 419 were significantly modulated by paraquat exposure. Biological pathway enrichment revealed the ubiquinone metabolism, a pathway directly linked to mitochondrial complex I proteins, confirming the well-known mechanism of PQ inducing oxidative stress. Additionally, this study also described the cholesterol biosynthesis modulation on HBMECs not yet described. In conclusion, our data indicate the toxic effect of PQ on HBMECs by downregulating proteins involved in mitochondrial complex I and cholesterol pathways.