K-ras is a well-studied oncogene and is commonly mutated in pancreas, lung and colorectal cancers. Cancers harboring K-ras mutations are difficult to treat due to drug resistance and metastasis properties. Cancer stem cells (CSCs) are considered a major cause of chemotherapeutic resistance and are thought to be responsible for tumor recurrence after therapy. K-ras mutations in CSCs are not well-understood. Here we demonstrate that K-ras mutations are related to CSCs' abilities to form tumor spheres and proliferate. We compared two colon cancer cell lines, HCT-116 and HT-29, the former is a K-ras mutated cell line and the latter is wild type. We found that HCT-116 cells treated with a K-rasG12C inhibitor S7333 formed significantly more tumor spheres than untreated cells (P=0.0474), while HT-29 (wild type) remained unchanged. However, the size of spheres (cell numbers per sphere) with S7333 treatment was smaller than the untreated cells, indicating their proliferation was inhibited. Consistent with this result, real time qRT-PCR showed that treated HCT-116 cells had lower Ki67 proliferation marker expression than untreated cells. Expression of Kras and self-renewal Lgr5 genes were significantly decreased, inversely TGF-beta1 was increased in the treated HCT-116 cells. Further, flow cytometry indicated that expression of CD133 increased and CD44 decreased in treated HCT-116 cell compared with HT-29 cells. These finding reveal that K-ras mutations are related to stem cell properties in colon cancer, and may therefore contribute to colorectal tumorigenesis and metastasis.