The pathogenesis of the marked pulmonary microvasculature injury, a distinguishing feature of COVID-19 acute respiratory distress syndrome (COVID-ARDS), remains unclear. Implicated in the pathophysiology of diverse diseases characterized by endothelial damage, including ARDS and ischemic cardiovascular disease, ceramide and in particular palmitoyl ceramide (C16:0-ceramide) may be involved in the microvascular injury in COVID-19. Using deidentified plasma and lung samples from COVID-19 patients, ceramide profiling by mass spectrometry was performed. Compared with healthy individuals, a specific 3-fold C16:0-ceramide elevation in COVID-19 patient plasma was identified. Compared with age-matched controls, autopsied lungs of individuals succumbing to COVID-ARDS displayed a massive 9-fold C16:0-ceramide elevation and exhibited a previously unrecognized microvascular ceramide-staining pattern and markedly enhanced apoptosis. In COVID-19 plasma and lungs, the C16-ceramide/C24-ceramide ratios were increased and reversed, respectively, consistent with increased risk of vascular injury. Indeed, exposure of primary human lung microvascular endothelial cell monolayers to C16:0-ceramide–rich plasma lipid extracts from COVID-19, but not healthy, individuals led to a significant decrease in endothelial barrier function. This effect was phenocopied by spiking healthy plasma lipid extracts with synthetic C16:0-ceramide and was inhibited by treatment with ceramide-neutralizing monoclonal antibody or single-chain variable fragment. These results indicate that C16:0-ceramide may be implicated in the vascular injury associated with COVID-19.