MicroRNAs (miRNAs) appear as crucial regulators of development in many organisms and are themselves highly regulated. In the silkworm, Bombyx mori, expression pattern of let-7 miRNA mimics the insect steroid-hormone (ecdysone) peaks and disruption of its expression affects the developmental transitions such as molting and metamorphosis. To understand regulatory mechanisms of the ecdysone induced expression of let-7 miRNA in the silkworm, we investigated the effects of knockdowns of ecdysone-responsive genes on let-7 miRNA expression using cultured cells derived from the silkworm ovaries (BmN cells). The expression level of let-7 miRNA in cells treated with Ponasterone A (an ecdysone analog) was two-fold higher than that in mock-treated cells. The same treatment dramatically increased the mRNA levels of ecdysone induced protein 75 (E75) A and B isoforms. RNAi-mediated knockdown of E75 gene repressed the Ponasterone A induced expression of let-7 miRNA. We found that the intron 2 of the putative let-7 encoding gene (KAIKOGA027151) had at least three putative monomeric response elements (MRE), which is a known binding site for E75A. Our reporter gene assays demonstrated that 1454 bp intronic sequence containing MRE1 and 2 was sufficient for the Ponasterone A induced expression. These results suggest that E75 regulates the ecdysone-induced expression of let-7 miRNA in the silkworm.