Significance Ubiquitination is a vital posttranslational modification in eukaryotes. A variety of microbial pathogens exploit this pathway during their infection. Legionella pneumophila , the causative bacterial pathogen of Legionnaires’ disease, has been shown to hijack host ubiquitination pathway via a large number of effectors. Recent studies revealed a family of effectors catalyzing a type of ubiquitin (Ub)-dependent posttranslational modification, namely PR-ubiquitination. Here we report 2 players, DupA and DupB, involved in this unconventional pathway. We found that DupA and DupB function as PR-Ub–specific deubiquitinases and play a role in regulating the PR-ubiquitination levels of host targets. Our results not only provide an expanding view of the PR-ubiquitination pathway, but also may facilitate the future identification of PR-ubiquitination pathways in eukaryotes.