The trophoblast (TR) is the first to differentiate during mammalian embryogenesis and play a pivotal role in the development of the placenta. We used a dual inhibitor system (PD0325901 and CHIR99021) with mixed feeders to successfully obtain bovine trophoblast stem-like (bTS) cells, which were similar in phenotype to mouse trophoblast stem cells (TSCs). The bTS cells that were generated using this system continually proliferated, displayed a normal diploid karyotype, and had no signs of altered morphology or differentiation even after 150 passages. These cells exhibited alkaline phosphatase (AP) activity and expressed pluripotency markers, such as OCT4, NANOG, SOX2, SSEA-1, SSEA-4, TRA-1-60, and TRA-1-81, and TR lineage markers such as CDX2, as determined by both immunofluorescence and reverse transcription-polymerase chain reaction (RT-PCR). Additionally, these cells generated dome-like structures, formed teratomas when injected into NOD-SCID mice, and differentiated into placenta TR cells in vitro. The microarray analysis of bTS cells showed high expression levels of many TR markers, such as TEAD4, EOMES, GATA3, ETS2, TFAP2A, ELF5, SMARCA4 (BRG1), CDH3, MASH2, HSD17B1, CYP11A1, PPARG, ID2, GCM1, HAND1, TDK, PAG, IFN-tau, and THAP11. The expression of many pluripotency markers, such as OCT4, SOX2, NANOG, and GDF3, was lower in bTS cells compared with in vitro-produced blastocysts; however, compared with bovine fetal fibroblasts, the expression of these pluripotency markers was elevated in bTS cells. The DNA methylation status of the promoter regions of OCT4, NANOG, and SOX2 was investigated, which were significantly higher in bTS cells (OCT4 23.90 , NANOG 74.40 , and SOX2 8.50 ) compared with blastocysts (OCT4 8.90 , NANOG 34.4 , and SOX2 3.80 ). In contrast, two promoter regions of CDX2 were hypomethylated in bTS cells (13.80 and 3.90 ) compared with blastocysts (18.80 and 9.10 ). The TSC lines that were established in this study may be used either for basic research that is focused on peri-implantation and placenta development or as donor cells for transgenic animal production.