Molting in insects is regulated by molting hormones (ecdysteroids), which are also crucial to insect growth, development, and reproduction etc. The decreased ecdysteroid in titre results from enhanced ecdysteroid inactivation reactions including the formation of 3‐epiecdyson under ecdysone oxidase and 3‐dehydroecdysone 3α‐reductase (3DE 3α‐reductase). In this paper, we cloned and characterized 3‐dehydroecdysone 3α‐reductase (3DE 3α‐reductase) in different tissues and developing stage of the silkworm, Bombyx mori L. The B. mori 3DE 3α‐reductase cDNA contains an ORF 783 bp and the deduced protein sequence containing 260 amino acid residues. Analysis showed the deduced 3DE 3α‐reductase belongs to SDR family, which has the NAD(P)‐binding domain. Using the Escherichia coli, a high level expression of a fusion polypeptide band of approx. 33 kDa was observed. High transcription of 3DE 3α‐reductase was mainly presented in the midgut and hemolymph in the third day of fifth instar larvae in silkworm. The expression of 3DE 3α‐reductase at different stages of larval showed that the activity in the early instar was high, and then reduced in late instar. This is parallel to the changes of molting hormone titer in larval. 3DE 3α‐reductase is key enzyme in inactivation path of ecdysteroid. The data elucidate the regulation of 3DE 3α‐reductase in ecdyteroid titer of its targeting organs and the relationship between the enzyme and metamorphosis.