Retrons mediate bacterial anti-phage defense by reverse transcribing non-coding RNA into multicopy single-stranded DNA (msDNA). Our study reveals the unique trimeric nucleoprotein structure of Eco2, which solely relies on a single protein for defense, unlike most other retrons. This structure supports an msDNA-dependent regulation of the Eco2's reverse-transcriptase and TOPRIM/RNase H (TR) fusion protein. We also show that Eco2's broad defense against various phages is triggered by a phage-encoded endonuclease that degrades the msDNA. msDNA decay in turn activates the TR domain for tRNA cleavage, resulting in shutdown of gene expression for abortive infection. Our findings not only advance the understanding of Eco2’s biogenesis and defense mechanism but also provide a structural basis for engineering of this system.