Invasion and metastasis are the major cause of deaths in patients with esophageal cancer. In this study, we isolated cancer stem-like cells from an esophageal squamous cell carcinoma cell line EC109 based on aldehyde dehydrogenase 1A1 (ALDH1A1), and found that ALDH1A1(high) cells possessed the capacities of self-renewal, differentiation and tumor initiation, indications of stem cell properties. To support their stemness, ALDH1A1(high) cells exhibited increased potential of invasion and metastasis as compared with ALDH1A1(low) cells. ALDH1A1(high) esophageal squamous cell carcinoma cells expressed increased levels of mRNA for vimentin, matrix metalloproteinase 2, 7 and 9 (MMP2, MMP7 and MMP9), but decreased the level of E-cadherin mRNA, suggesting that epithelial-mesenchymal transition and secretary MMPs may be attributed to the high invasive and metastatic capabilities of ALDH1A1(high) cells. Furthermore, we examined esophageal squamous cell carcinoma specimens from 165 patients and found that ALDH1A1(high) cells were associated with esophageal squamous dysplasia and the grades, differentiation and invasion depth, lymph node metastasis and UICC stage of esophageal squamous cell carcinoma, as well as poor prognosis of patients. Our results provide the strong evidence that ALDH1A1(high) cancer stem-like cells contribute to the invasion, metastasis and poor outcome of human esophageal squamous cell carcinoma.