RtcB enzymes comprise a widely distributed family of manganese- and GTP-dependent RNA repair enzymes that join 2',3'-cyclic phosphate ends to 5'-OH ends via RtcB-(histidinyl-N)-GMP, RNA 3'-phosphate, and RNA3'pp5'G intermediates. RtcB can ligate either 5'-OH RNA or 5'-OH DNA strands in vitro. The nucleic acid contacts of RtcB are uncharted. Here we report a 2.7 Å crystal structure of RtcB in complex with a 6-mer 5'-OH DNA oligonucleotide ApTpGpTpCpC, which reveals enzymic contacts of Asn202 to the terminal 5'-OH nucleophile; Arg238 to the ApT and TpG phosphates; Arg190 and Gln194 to the TpG phosphate; and an Arg190 π-cation interaction with the G3 nucleobase. The structural insights affirm functional studies of RtcB that implicated the conserved counterpart of Arg238 in engagement of the 5'-OH strand for ligation. The essential active site Cys98 that coordinates two manganese ions is oxidized to cysteine sulfonic acid in our structure, raising the prospect that RtcB activity might be sensitive to modulation during oxidative stress.