To explore the structural characteristics of gene promoter Oct4 in sheep, 5' upstream regulatory regions of ovine gene Oct4 were cloned using PCR, and the comparative alignment analysis among sheep, cow and pig was conducted as well. The results showed that the promoter was 2 951 bp in length. There were four conserved regions (CR 1 to 4) in the upstream regulatory sequence of gene Oct4 in sheep, cow and pig, and the four regions presented in high homology among the three species. A putative Sp1/Sp3 binding site and the overlapping hormone responsive element (HRE) in CR 1 region were identical to each other among the three species, moreover, there were rich in GC and a high number of CCC (A/T) CCC motifs in the upstream regions. The research could provide reference for futher understanding the gene Oct4 and facilitate the relevant research on sheep.