MicroRNAs (miRNA) regulate multiple physiological processes including development and metamorphosis in insects. In the current study, we demonstrate that a conserved invertebrate miRNA-14 (miR-14) plays an important role in ecdysteroid regulated development in the silkworm Bombyx mori, a lepidopteran model insect. Ubiquitous transgenic overexpression of miR-14 using the GAL4/UAS system resulted in delayed silkworm larval development and smaller body size of larva and pupa with decrease in ecdysteriod titers. On the contrary, miR-14 disruption using the transgenic CRISPR/Cas9 system led to a precocious wandering stage with increase in ecdysteriod titers. We identified that the hormone receptor E75 (E75) and the ecdysone receptor isoform B (ECR-B), which both serve as essential mediators in the ecdysone signaling pathway, as putative target genes of miR-14 by in silico target prediction. Dual-luciferase reporter assays confirmed the binding of miR-14 to the 3'UTRs of E75 and ECR-B in a mammalian HEK293T cell line. Furthermore, transcription levels of E75 and ECR-B were significantly affected in both miR-14 overexpression and knockout transgenic animals. Taken together, our data suggested that the canonical invertebrate miR-14 is a general regulator in maintaining ecdysone homeostasis for normal development and metamorphosis in B. mori.