Non-CG methylation has been associated with stemness regulation in embryonic stem cells. By comparing differentially expressed genes affected by non-CG methylation between tumour and corresponding non-tumour tissues in oesophageal squamous cell carcinoma (OSCC), we find that Integrin α7 (ITGA7) is characterized as a potential cancer stem cell (CSC) marker. Clinical data show that a high frequency of ITGA7+ cells in OSCC tissues is significantly associated with poor differentiation, lymph node metastasis and worse prognosis. Functional studies demonstrate that both sorted ITGA7+ cells and ITGA7 overexpressing cells display enhanced stemness features, including elevated expression of stemness-associated genes and epithelial-mesenchymal transition features, as well as increased abilities to self-renew, differentiate and resist chemotherapy. Mechanistic studies find that ITGA7 regulates CSC properties through the activation of the FAK-mediated signalling pathways. As knockdown of ITGA7 can effectively reduce the stemness of OSCC cells, ITGA7 could be a potential therapeutic target in OSCC treatment.