Purpose Metastasis and drug resistance are the major limitations in the survival and management of cancer patients. This study aimed to identify the mechanisms underlying HT29 colon cancer cell chemoresistance acquired after sequential exposure to 5-fluorouracil (5FU), a classical anticancer drug for treatment of epithelial solid tumors. We examined its clinical relevance in a cohort of colon cancer patients with liver metastases after 5FU-based neoadjuvant chemotherapy and surgery. Results We show that a clonal 5F31 cell population, resistant to 1μM 5FU, express a typical cancer stem cell-like phenotype and enter into a reversible quiescent G0-state upon re-exposure to higher 5FU concentrations. These quiescent cells overexpressed the tyrosine kinase c-Yes that became activated and membrane-associated upon 5FU exposure. This enhanced signaling pathway induced the dissociation of the Yes/YAP (Yes-associated protein) molecular complex and depleted nuclear YAP levels. Consistently, c-Yes silencing decreased nuclear YAP accumulation and induced cellular quiescence in 5F31 cells cultured in 5FU-free medium. Importantly, c-Yes and YAP transcript levels were higher in liver metastases of colon cancer patients after 5FU-based neoadjuvant chemotherapy. Moreover, the c-Yes and YAP levels positively correlated with colon cancer relapse and shorter patient survival (p<0.05 and p<0.025, respectively). Conclusions We identified c-Yes and YAP as potential molecular targets to eradicate quiescent cancer cells and dormant micrometastases during 5FU chemotherapy and resistance and as predictive survival markers for colon cancer.