Host cells contest invasion by intracellular bacterial pathogens with multiple strategies that recognise and / or damage the bacterial surface. To identify novel host defence factors targeted to intracellular bacteria, we developed a versatile proximity biotinylation approach coupled to quantitative mass spectrometry that maps the host-bacterial interface during infection. Using this method, we discovered that intracellular Shigella and Salmonella become targeted by UFM1-protein ligase 1 (UFL1), an E3 ligase that catalyses the covalent attachment of Ubiquitin-fold modifier 1 (UFM1) to target substrates in a process called UFMylation. We show that Shigella antagonises UFMylation in a dual manner: first, using its lipopolysaccharide (LPS) to shield from UFL1 recruitment; second, preventing UFM1 decoration by the bacterial effector IpaH9.8. Absence of UFMylation leads to an increase of bacterial burden in both human cells and zebrafish larvae, suggesting that UFMylation is a highly conserved antibacterial pathway. Contrary to canonical ubiquitylation, the protective role of UFMylation is independent of autophagy. Here, we used an proteomics-based approach to host effector binding to Shigella in human infected cells. This allowed to confirm UFM1 recruitment to Shigella surface and to identify host effector binding to Shigella in a UFM1-dependent matter. This result reveals potential UFM1 protein substrates on the surface of Shigella in infetec human cells. To do so, we performed siRNA knockdown against UFM1 in HeLa cells prior to infection with the hyperinvasive Shigella flexneri afaI strain considered as the wild-type strain. Alternatively, HeLa cells were infected with S. flexneri ΔrfaCΔipaH9.8 considered as mutant. There were four biological groups with each four replicates: HeLa cells with siUFM1 with wild-type S. flexneri, HeLa cells with non-targeting siRNA with wild-type S. flexneri, siUFM1 with Shigella mutant and non-targeting siRNA with Shigella mutant.