To deeply investigate the effect of insulin-like androgenic hormone (IAG) on the regulation of gene expression in shrimp Litopenaeus vannamei, we conducted in vivo IAG knockdown and recombinant protein treatment experiments. By setting four induction temperatures, 16, 20, 28 and 37 ℃, we investigated the influence of temperature on the prokaryotic expression efficiency of the IAG recombinant protein. The findings showed that induction at 28 ℃ for 12 hours resulted in a relatively high yield of pET32a-IAG recombinant protein in the inclusion bodies of the prokaryotic expression system. In the IAG dsRNA short-term interference experiment, IAG expression was significantly reduced by 97%. Meanwhile, transcriptome analysis of the androgenic gland showed a significant up-regulation of the expression of the reproduction-related gene CYP2L1, as well as the arachidonic acid pathway gene GPx, while the expression of the metabolism-related gene TnC was significantly down-regulated (P < 0.05). The results of this study suggested that IAG may play an important regulatory role in the process of reproduction and development of L. vannamei, and that changes in its expression level can significantly affect the expression pattern of reproduction-related and metabolism-related genes, which in turn may influence the process of reproduction and development.