Significance Shigella flexneri , a deleterious bacterium, causes massive human infection cases and deaths worldwide. To facilitate survival and replication in infected host cells, S. flexneri can secrete two highly similar E3 ligase effectors, IpaH1.4 and IpaH2.5, to subvert the linear ubiquitin chain assembly complex (LUBAC), a key player involved in numerous antibacterial signaling pathways of host cells but with poorly understood mechanisms. In this study, through systematic biochemical and structural characterization, we elucidate the multiple tactics adopted by IpaH1.4/2.5 to disarm the human LUBAC and provide mechanistic insights into the subversion of host LUBAC by IpaH1.4/2.5 of S. flexneri .