Cytosine modifications expand the information content of genomic DNA in both eukaryotes and prokaryotes, providing means for epigenetic regulation and self versus nonself discrimination. For example, the methyl‐directed restriction endonuclease, McrBC, recognizes and cuts invading bacteriophage DNA containing 5‐methylcytosine (5mC), 5‐hydroxymethylcytosine (5hmC), and N4‐methylcytosine (4mC), leaving the unmodified host DNA intact. Here, we present cocrystal structures of McrB‐N bound to DNA oligoduplexes containing 5hmC, 5‐formylcytosine (5fC), and 4mC, and characterize the relative affinity of McrB‐N to various cytosine variants. We find that McrB‐N flips out modified bases into a protein pocket and binds cytosine derivatives in the order of descending affinity: 4mC > 5mC > 5hmC ≫ 5fC. We also show that pocket mutations alter the relative preference of McrB‐N to 5mC, 5hmC, and 4mC
