Despite numerous reported attempts, successful isolation of genuine bovine embryonic stem cells has been rare. Previous studies have shown that Thiazovivin, a Rho-associated kinase inhibitor, improves the survival and self-renewal of human embryonic stem cells. The present study demonstrated the effect of Thiazovivin on the derivation of bovine embryo-derived pluripotent stem cells. The attachment rates of blastocyst and embryonic cell clumps onto feeder cells in the Thiazovivin treatment group were significantly higher than those of the control. The pluripotency markers of OCT4 and NANOG, and the adhesion molecule E-cadherin were increased by Thiazovivin treatment. This study suggests that Thiazovivin treatment improves the maintenance of stemness in a putative embryo-derived pluripotent stem cells population by promoting the expression of pluripotency marker genes as well as enhancing the expression of E-cadherin resulting in an increase in cell adhesion. This study was supported by a grant from the National Research Foundation of Korea (NRF-2006-2004042, and no. 2014050477 through the Oromaxillofacial Dysfunction Research Center for the Elderly at Seoul National University) and the Technology Development Program for Agriculture and Forestry, Ministry of Agriculture, Food and Rural Affairs (MAFRA; 111160-04), Republic of Korea