Endonuclease G (EndoG) is a conserved endonuclease implicated in mitochondrial DNA (mtDNA) replication, maintenance of mtDNA integrity under oxidative stress, and the removal of nuclear and paternal mtDNA during apoptosis and early embryogenesis. Despite its biological significance, the substrates targeted by EndoG and its cleavage preferences remain unclear. Here, we characterize human EndoG (hEndoG) across diverse nucleic acid substrates, including single-stranded DNA (ssDNA), double-stranded