Bovine embryonic stem cells (bESCs) have not been successfully established yet. One reason could be that CDX2, as the trophectoderm regulator, expresses in bovine inner cell mass (ICM), which probably becomes a technical barrier for maintaining the pluripotency of bESCs in vitro. We hypothesized that CDX2 knockdown (CDX2-KD) could remove such negative effort, which will be helpful for capturing complete and permanent capacity of pluripotency. Expression and localization of pluripotent genes were not affected in CDX2-KD blastocysts. The CDX2-KD bESCs grew into monolayers on feeder layer. Pluripotent genes expressed at an improved levels and lasted longer time in CDX2-KD bESCs, along with down-regulation of DNA methylation on promoters of both OCT4 and SOX2. The cystic structure typical for trophoblast cells did not show during culturing CDX2-KD bESCs. CDX2-KD bESC-derived Embryoid bodies showed with compact morphology and with the improved levels of differentiations in three germ layers. CDX2-KD bESCs still carried the capacity of forming teratomas with three germ layers after long-term culture. In summary, CDX2 in bovine ICM was inducer of trophoblast lineage with negative effect on maintenance of pluripotency of bESCs. Precise regulation CDX2 expression to switch on/off will be studied next for application on establishment of bESCs.