Cancer stem-like cells exist in many malignancies and several stem cell-related genes and microRNAs, such as Bmi-1 and miR-203, have been identified as cancer stem-like cell regulators using gene microarray or sequencing analysis. Previously, we used side population (SP) sorting to enrich cancer stem-like cells from esophageal squamous cell carcinoma (ESCC) cell line EC9706. Our results demonstrated that EC9706 SP cells shared common features of cancer stem-like cells. In this study, we examined the expression of Bmi-1 and miR-203 in ESCC SP and non-SP (NSP) cells. Our results showed that, when compared with NSP cells, Bmi-1 was up-regulated and miR-203 was down-regulated in SP cells. During the differentiation from SP to NSP cells, the expression levels of Bmi-1 were gradually decreased. Overexpression of miR-203 resulted in a significant reduction of endogenous Bmi-1 protein level in EC9706 cells. SP and NSP analyses revealed that the SP cell fraction was markedly decreased in miR-203 overexpressed cells. miR-203 overexpressed cells also showed a significant reduction in colony formation, which was resistant to chemotherapeutic drug treatment and tumorigenicity in nude mice. Rescue experiments demonstrated that ectopic expression of Bmi-1 in miR-203 overexpressed cells increased the SP fraction and restored cell proliferation. Taken together, these results indicated that stem renewal factor Bmi-1 was a direct target of miR-203. The regulation of Bmi-1 by miR-203 may play an important role in controlling cell proliferation and self-renewal of esophageal cancer stem-like cells. It may also promote the development of new therapeutic strategies and efficient drugs that target ESCC stem-like cells.