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The enzyme 5,10‐methylenetetrahydrofolate dehydrogenase (MTHFD) is essential for the production of certain amino acids (glycine, serine, and methionine) and nucleic acids (thymidylate and purine). Here, we identified a cDNA encoding this enzyme from the silkworm Bombyx mori. The recombinant B. mori MTHFD (bmMTHFD) expressed in Escherichia coli recognized 5,10‐methylenetetrahydrofolate and 5,10‐methenyltetrahydrofolate as substrate in the presence of NADP+ as well as NAD+. The bmMTHFD structure was determined at a resolution of 1.75 Å by X‐ray crystallography. Site‐directed mutagenesis indicated that the amino acid residue Tyr49 contributed to its catalytic activity. Our findings provide insight into the mechanism underlying the activity of MTHFD from B. mori and potentially other insects and may therefore facilitate the development of inhibitors specific to MTHFD as insecticides. Here, we cloned cDNA encoding Bombyx mori 5,10‐methylenetetrahydrofolate dehydrogenase (bmMTHFD). Recombinant bmMTHFD produced in bacteria recognized MTHF and 5,10‐methenyltetrahydrofolate as substrate in the presence of NADP+ as well as NAD+. The structure of bmMTHFD was determined at 1.75 Å by X‐ray crystallography, and we found that Ser46, Tyr49, Lys53, Gln97, and Asp120 contribute to catalysis. [ABSTRACT FROM AUTHOR]