BACKGROUNDReproductive system plays a crucial role in insect survival, reproduction and species specificity. Understanding the molecular mechanisms underlying reproductive organogenesis contributes to improving the efficiency of sterile insect technique marked by an eco‐friendly pest management strategy. Lepidoptera is one of the largest orders of insects, most of which are major pests in agriculture and forestry. Our study aimed to screen the genes responsible for reproductive organogenesis and unravel the mechanism underlying female reproductive organ defects.RESULTSMorphological investigation of female reproductive organs showed a defective connection between oviductus geminus and oviductus communis on the 2nd day of pupa (P2) in Speckled mutant silkworm. RNA_Seq identified a total of 18,049 transcripts that were expressed in the P2 female internal reproductive organs without ovary in Spc/+ compared to +Spc/+Spc. Differential expression analysis identified 312 up‐regulated genes and 221 down‐regulated genes in Spc/+. KEGG analysis identified 44 significantly enriched pathways. The results of qRT‐PCR performed on 33 genes significantly matched the outcomes of the RNA_Seq. Dysfunction of Cyclin B3 resulted in a defective connection of the oviductus communis with the ovariole, dysfunction of oogenesis, and a petite body. Moreover, homozygous recessive lethality of Cyclin B3/Cyclin B3 occurred during early embryogenesis.CONCLUSIONOur results suggested that Cyclin B3 is a pleiotropic functional gene that regulates early embryogenesis, oogenesis, development, and female reproductive organogenesis. These results showed that Cyclin B3 has significant effects on lepidopteran mortality, growth, and reproductive physiology, which might be considered a novel and potentially eco‐friendly target for lepidopteran pest management.This article is protected by copyright. All rights reserved.