Cancer cell molecular mimicry of stem cells (SC) follows with enhanced proliferative and renewal capacities. In support, numerous mediators of SC self-renewal have been evinced to exhibit oncogenic potential. More and more researches showed that the embryonic stem cell self-renewal genes express in various cancer cells. In this study, we sought to test the tumorigenic functions of NANOG, particularly, in esophageal cancer (EC). Using quantitative RT-PCR and western blotting, we confirmed that EC cells highly express NANOG mRNA and protein. We then constructed a shRNA-mediated plasmid to knockdown of NANOG mRNA. We observed that NANOG deficiency in Eca109 cells decreased clone formation, cell proliferation, and showed G1 arrest. To further investigate the functions and mechanisms of NANOG in Eca109 cells, we detected the changes of multiple signaling molecules when NANOG deficiency. We foud that NANOG deficiency affected multiple genes, particularly, supressed drug-resistance via down-regulated ABCG2 in Eca109 cells, and caused G1 arrest by down-regulated cyclin D1 (CCND1) expression. The present loss-of-function work, establish the integral role for NANOG in Eca109 cell proliferation, drug resistance, and shed light on its mechanisms of action.