DNA nucleases catalyze the cleavage of phosphodiester bonds. These enzymes play crucial roles in various DNA repair processes, which involve DNA replication, base excision repair, nucleotide excision repair, mismatch repair, and double strand break repair. In recent years, new nucleases involved in various DNA repair processes have been reported, including the Mus81 : Mms4 (Eme1) complex, which functions during the meiotic phase and the Artemis : DNA-PK complex, which processes a V(D)J recombination intermediate. Defects of these nucleases cause genetic instability or severe immunodeficiency. Thus, structural biology on various nuclease actions is essential for the elucidation of the molecular mechanism of complex DNA repair machinery. Three-dimensional structural information of nucleases is also rapidly accumulating, thus providing important insights into the molecular architectures, as well as the DNA recognition and cleavage mechanisms. This review focuses on the three-dimensional structure-function relationships of nucleases crucial for DNA repair processes.