The　Holliday　junction,　a　four-way　DNA　structure,　is　an　important　intermediate　of　homologous　recombination.　Proper　Holliday　junction　resolution　is　critical　to　complete　the　recombination　process.　In　most　bacterial　cells,　the　Holliday　junction　cleavage　is　mainly　performed　by　a　specific　endonuclease　RuvC.　Here,　we　describe　the　biochemical　properties　and　the　crystal　structure　of　RuvC　from　an　opportunistic　pathogen,　Pseudomonas　aeruginosa　(PaRuvC).　PaRuvC　specifically　binds　to　the　Holliday　junction　DNA　and　preferentially　cleaves　it　at　the　consensus　5′-TTC-3′.　PaRuvC　uses　Mg2+　as　the　preferred　divalent　metal　cofactor　for　Holliday　junction　cleavage　and　its　optimum　pH　is　8.0–9.0.　Elevated　temperatures　(37–60　°C)　boost　the　catalytic　activity,　but　temperatures　higher　than　53　°C　reduce　the　protein　stability.　The　crystal　structure　of　PaRuvC　determined　at　2.4　Å　and　mutagenesis　analysis　reveal　key　residues　involved　in　the　dimer　formation,　substrate　binding　and　catalysis.　Our　results　are　expected　to　provide　useful　information　to　combat　antibiotic　resistance　of　Pseudomonas　aeruginosa　by　targeting　its　homologous　recombination　system.　©　2020　Elsevier　Inc.