E75 is an important regulator of the ecdysone signaling pathway in shrimp. In order to gain insight into the structure and function of this gene, we screened and analyzed all E75 gene transcripts based on the transcriptome and genome data of Litopenaeus vannamei, and identified 6 alternatively spliced LvE75 isoforms (named LvE75-1, LvE75-2, LvE75-3, LvE75-4, LvE75-5 and LvE75-6). LvE75-1/2/4/5/6 contained DBD and LBD domains consistent with E75A/C of Drosophila melanogaster, and LvE75-3 only had LBD domains consistent with Drosophila E75D. During molting process, LvE75s were highly expressed in D3–D4 stage. In adult tissues, various isoforms of LvE75 were expressed in all tissues and were highly expressed in the epidermis, gut and gill, with only LvE75-3 being highly expressed in hepatopancreas, blood cells and lymphoid tissues. According to the conserved domain sequence, we designed primers for double-stranded RNA (dsRNA) interference. In LvE75 gene RNAi samples, qRT-PCR results showed that spo, phm and dib were down-regulated, and shd was up-regulated, indicating that LvE75 might regulate Halloween genes to influence ecdysone synthesis. Br-C and Ftz-f1 were down-regulated and HR3 was up-regulated after LvE75 were interfered, indicating LvE75 might influence the downstream early response genes of ecdysone signal pathway. After continued interference in LvE75 for 12 days, the molting rate was significantly lower compared with control group, but the death rate of dsLvE75 group was significantly higher, suggesting LvE75 gene played an important role in shrimp molting and survival.