Abstract Background: The potentiation and activation of Wnt signaling pathways are now assumed to mediate the self-renewal and proliferation of colon cancer stem cells that are responsible for therapeutic resistance, tumor relapse, and metastasis. Aim: We aimed to evaluate LGR5, Prox1, and Notch1 immunohistochemical expression in stage II to III colon cancer. Their predictive role of tumor relapse, overall survival, and disease-free survival was statistically analyzed. Results: Our results revealed that high LGR5 expression was identified in 56.7% of the patients, LGR5 expression was significantly associated with left-sided tumors (P<0.001). Moreover, its expression was significantly associated with the unfavorable tumor characteristics including high grade, deep invasion (pT), lymph node metastasis, and advanced tumor stage (P<0.001 for each). High Prox1 expression was observed in 65% of the cases, and its expression was significantly associated with tumor grade, lymph node metastasis, and the advanced tumor stage (P=0.004, 0.009, 0.016, respectively). Positive Notch1 expression was identified in 35% of patients, and it was inversely associated with high grade lymph node metastasis, deep invasion (pT), and advanced tumor stage (P<0.001 for each). During the follow-up period, the tumor relapse was significantly associated with high LGR5, high Prox1, and negative Notch1 expression. Shorter overall survival and disease-free survival were significantly associated with high LGR5, high Prox1, and negative Notch1 expression. Conclusions: High LGR5, high Prox1, and negative Notch1 expression are unfavorable prognostic factors in colon cancer. Prox1 is a crucial regulator of Notch-independent LGR5+ stem cells that is mostly responsible for relapse and therapeutic resistance in stage II to III colon cancer. Key Words: colon cancer, LGR5, Prox1, Notch1, immunohistochemistry, cancer colon