Insect development and metamorphosis are regulated by the coordination of ecdysone and juvenile hormones. Insect microRNAs (miRNAs) also act in insect development and metamorphosis by regulating genes in the ecdysone cascade. Although hundreds of insect miRNAs have been identified, the physiological functions of most remain poorly understood. Here, we report that a conserved insect miRNA, microRNA-281 (miR-281), regulates the ecdysone receptor (EcR), in an isoform-specific manner in the silkworm Bombyx mori. The B. mori EcR (BmEcR) gene encodes three isoforms: BmEcR-A, BmEcR-B1 and BmEcR-B2. The 3'UTR regions of A and B genes, which contain multiple potential microRNA targeting sites, are distinct. Target prediction revealed that miR-281 may specifically target the 3'UTR of BmEcR-B. Using a dual luciferase reporter assay in HEK293T cells, we confirmed that miR-281 suppressed transcription of BmEcR-B but not BmEcR-A. The expression of miR-281 and BmEcR-B are well coordinated in the Malpighian tubules from the fourth larval molt to pupation. In the Malpighian tubules of fifth instar larvae, BmEcR-B protein expression was down-regulated after injection of a miR-281 mimic while up-regulated after injection of a miR-281 inhibitor. miR-281 expression was suppressed by 20-hydroxyecdysone treatments but not affected by juvenile hormone treatments. Based on these findings, we propose that miR-281 participates in B. mori developmental regulation in the Malpighian tubules through suppression of BmEcR-B expression.