Updated project metadata. The drivers of tissue necrosis in Mycobacterium ulcerans infection (Buruli ulcer disease) have historically been ascribed solely to the cytotoxic action of the diffusible endotoxin, mycolactone. However, clinical evidence supporting a strong vascular contribution to disease aetiology has, until recently, been under-explored. We have now dissected mycolactone’s effects on primary vascular endothelial cells in vitro and in vivo. We show that mycolactone’s effect on endothelial morphology, adhesion, migration, and permeability are dependent on its action at the Sec61 translocon. Unbiased quantitative proteomics identified a profound effect on proteoglycans, driven both by a rapid loss of type II transmembrane proteins of the Golgi, including enzymes required for glycosaminoglycan (GAG) synthesis and the core proteins themselves. Loss of the glycocalyx is likely to be of particular mechanistic importance, since knockdown of galactosyltransferase II (beta-1,3-galactotransferase 6; B3Galt6), the GAG linker-building enzyme, phenocopied the permeability and phenotypic changes induced by mycolactone. Additionally, mycolactone depleted many secreted basement membrane components and microvascular basement membranes were disrupted in vivo. Remarkably, exogenous addition of laminin-511 reduced cell rounding, restored cell attachment and reversed the defective migration caused by mycolactone suggesting that this might be a viable therapeutic avenue in the future.