MUS81　is　an　important　structure-specific　endonuclease　responsible　for　the　processing　of　stalled　replication　forks　and　recombination　intermediates.　In　human,　MUS81　functions　by　forming　complexes　with　its　regulatory　subunits　EME1　and　EME2,　playing　distinct　roles　in　G2/M　and　S　phases.　Although　the　structures　of　MUS81-EME1　have　been　intensively　studied,　there　is　no　structure　information　available　about　MUS81-EME2.　Here,　we　report　the　crystal　structure　of　MUS81-EME2,　which　reveals　an　overall　protein　fold　similar　to　that　of　MUS81-EME1　complex.　Further　biochemical　and　structural　characterization　shows　that　the　MUS81-EME1　and　MUS81-EME2　complexes　are　identical　in　substrate　recognition　and　endonuclease　activities　in　vitro,　implying　that　the　distinct　cellular　roles　of　the　two　complexes　could　arise　from　temporal　controls　in　cells.　Finally,　an　extensive　structure-guided　mutagenesis　analysis　provides　implications　for　the　molecular　basis　of　how　the　MUS81-EME　endonucleases　recognize　various　DNA　substrates　in　a　structure-selective　manner.