The RNA-guided endonuclease Cas9 has made genome editing a widely accessible technique. Similar to Cas9, endonucleases from theArgonaute protein family also use oligonucleotides as guides to degrade invasive genomes. Here we report that the Natronobacterium gregoryiArgonaute (NgAgo) is a DNA-guided endonuclease suitable for genome editing in human cells. NgAgo binds 5' phosphorylated single-stranded guide DNA (gDNA) of ～24 nucleotides, efficiently creates site-specific DNA double-strand breaks when loaded with the gDNA. The NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM), as does Cas9, and preliminary characterization suggests a low tolerance to guide-target mismatches and high efficiency in editing (G+C)-rich genomic targets.