Abstract: In this study, we cloned and sequenced the endogenous jaagsiekte sheep retrovirus（enJSRV） env gene, and using bioinformatics method to analyzed the similarity, genetic evolution, physicochemical properties, glycosylation sites, phosphorylation sites, B cell antigen epitopes and their secondary and tertiary structures of enJSRV Env protein. The complete enJSRV env gene sequence was obtained in the experiment, with a total length of 1 836 bp, encoding 611 amino acids. No exogenous exJSRV-specific "YXXM" motif was found in the TM region encoded by env gene, and env gene is a highly conserved gene. The similarity of the env gene sequence and the enJSRV env gene sequence registered in GenBank is 98.7%～99.5%, and the deduced amino acid sequence similarity is 98.0%～99.5%. The sequence similarity of exJSRV env gene was 88.3%～95.2% and that of the deduced amino acid sequence was 92.3%～93.8%. Bioinformatics analysis showed that enJSRV Env protein was a weak base, hydrophilic and unstable protein. In the secondary structure, α-helix and irregular crimps are the main structures, while the similarity modeling of the tertiary structure is successful, which mainly consists of helix and irregular crimps. The subcellular localization of Env protein was mainly distributed in the endoplasmic reticulum, and there was no signal peptide but existed transmembrane domain. There were 25 serine phosphorylation sites, 21 threonine phosphorylation sites, 5 tyrosine phosphorylation sites and 4 glycosylation sites in the protein sequence. The protein contained multiple B cell dominant epitopes and multiple dominant CTL epitopes. The results of this study provide a new idea for further elucidating the biological role of enJSRV Env protein in sheep.