Abstract 【Aim】 To search conserved miRNA target sites on the 3′UTRs of insect testis stem cell self-renewal associated genes, and to provide references for studying the miRNA-gene relationship in the process of insect spermatogenesis. 【Methods】 Testis stem cell self-renewal associated genes were identified in 27 holometabolan insects by BLAST search, and the 3′ UTRs of these genes were predicted. The miRNA target sites on the 3′UTRs of these genes were predicted by using TargetScan. The 3′UTRs of Chinmo and Imp containing let-7-5p target site of Bombyx mori and Plutella xylostella were cloned, and the target-gene relationship was verified by dual-luciferase reporter system. 【Results】 Seventeen testis stem cell self-renewal associated genes were identified in 27 holometabolan insects. By using TargetScan, 203 miRNA target sites were identified. A high number of conserved miRNA target sites were found on the 3′UTRs of Zfh-1, Chinmo, Ken and Imp. The number of conserved miRNA target sites was different among various insects. The let-7-5p target sites on Chinmo and Imp were conserved in insects and could be confirmed by other miRNA target prediction programs. The 3′UTRs of these two genes were cloned from B. mori and P. xylostella. The dual-luciferase reporter system in 293T cells validated the regulation of let-7-5p on Chinmo and Imp from these two insect species. 【Conclusion】 In this study, a large number of miRNA target sites were found on the 3′ UTRs of Zfh-1, Chinmo, Ken and Imp. The let-7-5p target sites on 3′UTRs of Chinmo and Imp are conserved in holometabolan insects. The target relationships between let-7-5p and Chinmo or Imp genes of B.mori and P. xylostella were verified by dual luciferase reporter system. This study will be helpful for better understanding the functions of miRNA in insect spermatogenesis.