Transfer RNA (tRNA) commonly undergoes multiple modifications essential for its role in intracellular translation. Modifying at position 34 is particularly important for accurate gene expression and cellular function. Gram-negative bacteria, such as Fusobacterium nucleatum (F. nucleatum), use a special pathway involving proteins such as carboxy-S-adenosyl-L-methionine synthase CmoA, carboxymethyltransferase CmoB, and 5-carboxymethyluridine methyltransferase CmoM to modify tRNA at this position. F. nucleatum is commonly found in the oral cavity and is associated with colorectal cancer progression. Therefore, studying its tRNA modification will help develop drugs to inhibit F. nucleatum, which highlights the importance of studying its tRNA modification mechanism. This study focuses on FnCmoM, a key enzyme in the tRNA modification pathway of F. nucleatum. We successfully expressed and purified FnCmoM and determined its crystal structure at 1.5 Å resolution using X-ray crystallography. Therefore, this study lays the foundation for understanding bacterial evolution and potential drug targets against F. nucleatum.