Background: Oleanolic acid (OA), a pentacyclic triterpenoid exhibiting specific anti-cancer properties and highly effective antioxidant activity, was isolated from traditional Chinese medicinal herbs. Conversely, the OA that impacts colon cancer (CC) cells and its underlying mechanisms remain poorly understood. Methods: The cytotoxic effect of OA alone or OA-5-Fluorouracil (5-FU) combination on normal and CC cells was analyzed by methyl thiazolyl diphenyl-tetrazolium bromide (MTT). Then, the impact of OA on CC cell lines (LoVo and HT-29) proliferation and stemness were measured using colon formation and tumorsphere formation assays. Octamerbinding transcription factor 4 (Oct4), Prominin-1 (CD133), Nanog, and transcription factor SOX-2 (SOX2) are cell stemness-related indicators whose expression was assessed using fluorescence qPCR assay, Western blotting, and immunohistochemistry. The effect of OA on the proliferative potency of CC cells was evaluated using an in vivo model. Results: The stem-like characteristics and clone production of colon cancer cells were markedly reduced by OA alone or in combination with OA-5-FU. Moreover, OA increases the susceptibility of CC cells to 5-FU by blocking the cell stemness-related markers (CD133, Nanog, SOX2, and Oct4) expression levels both in vitro and in vivo, as well as by inactivating the activator of transcription 3 (STAT3 signaling) and Janus kinase 2/signal transducer (JAK2). Conclusion: These findings imply that oleanolic acid, both in vitro and in vivo, suppresses the JAK2/STAT3 pathway, which in turn reverses chemoresistance and decreases colon cancer cell stemness. Therefore, by reducing the recommended amount of 5-FU, this strategy may improve chemotherapeutic effectiveness and minimize undesired side effects.