Renal cell carcinoma (RCC) is a deadly malignancy due to its tendency to metastasize and resistance to chemotherapy. Stem-like tumor cells often confer these aggressive behaviors. We discovered an endoglin (CD105)-expressing subpopulation in human RCC xenografts and patient samples with a greater capability to form spheres in vitro and tumors in mice at low dilutions than parental cells. Knockdown of CD105 by short hairpin RNA and CRISPR/cas9 reduced stemness markers and sphere-formation ability while accelerating senescence in vitro. Importantly, downregulation of CD105 significantly decreased the tumorigenicity and gemcitabine resistance. This loss of stem-like properties can be rescued by CDA, MYC, or NANOG, and CDA might act as a demethylase maintaining MYC and NANOG. In this study, we showed that Endoglin (CD105) expression not only demarcates a cancer stem cell subpopulation but also confers self-renewal ability and contributes to chemoresistance in RCC. •CD105+ cells sorted from kidney cancer cell line xenografts are stem-like cancer cells•CD105 demarcates a chemotherapy-resistant population in clear cell kidney cancer•CD105 mediates the self-renewal potential via CDA, MYC, and NANOG in ccRCC•CD105 knockdown abrogates chemoresistance in CD105+ ccRCC stem cells In this article, Xu, Wu, and colleagues show that CD105 not only demarcates the stem-like cancer subpopulation but also confers the self-renewal and gemcitabine chemoresistance through CDA, MYC, and NANOG in clear cell renal cell carcinoma (ccRCC). Downregulation of CD105 might serve as a ccRCC targeted therapy toward the stem-like cancer cell subpopulation.