Prokaryotic Argonautes (pAgos) form a diverse family of nucleic acid-guided proteins that defend their hosts against invading plasmid and viral DNA through various mechanisms. Various pAgos from the long-A clade recruit small single-stranded DNA guides to bind and cleave complementary invading DNA targets. However, the mechanism underlying DNA-specific catalytic activation has remained unclear. Using a combination of biochemical, structural, and single molecule studies, we reveal that the long-A pAgo from Clostridium butyricum (CbAgo) preferentially binds DNA over RNA, and that only DNA-guided target DNA binding induces CbAgo homodimerization. Homodimerization is essential for catalytic activation and for CbAgo-mediated defense against bacteriophages. Finally, we show that de novo designed CbAgo dimerization interface-binders inhibit CbAgo both in vitro and in vivo. Combined, our study reveals why long-A pAgo activity is DNA-specific, that their catalytic activation is controlled through target DNA binding-induced homodimerization, and that this control mechanism is broadly conserved among long-A pAgos.