Antibiotic resistance has become a serious threat to global health. Its rapid spread is causing the urgent need of novel antibiotics. Phospho-N-acetyl-muramoyl-pentapeptide translocase (MraY) belongs to the polyisoprenyl-phosphate N-acetylglucosaminosugar-1-phosphate (PNPT) superfamily and catalyses the first step in bacterial peptidoglycan synthesis pathway. MraY is the site of action for four classes of natural product nucleoside antibiotics (tunicamycins, ribosamino-uridines, uridylpeptides and capuramycins), hence favoring it as a promising target for the development of novel antibiotics. Here the X-ray structure of MraY in complex with the natural product nucleoside inhibitor tunicamycin is presented. The structure together with biophysical characterisation gives insights into molecular basis of tunicamycin binding and allows conclusions about tunicamycin inhibition of eukaryotic N-linked glycosylation.