Synergistic therapy is emerging as a promising strategy for improving the chemotherapeutic efficacy of anticancer drugs. Addition of adjuvants with standard anticancer drugs has shown successful reduction of adverse side effects. The synthetic drug 5-Fluorouracil (5-FU) shows several side effects upon prolonged chemotherapy, thereby restricting its long-term clinical application. Several studies have reported anticancer potential and anti-inflammatory activity of tangeretin (TAN) towards mammalian cells. Therefore, we investigate whether the combination of TAN with 5-FU increases their anticancer potential against colorectal cancer. In this study, we examined the synergistic activity of TAN and 5-FU on the viability of several human cancer and normal cells. Several possible mechanistic pathways were screened, and found that co-exposure of TAN and 5-FU accelerates oxidative-stress and increases endogenous-ROS generation, which sequentially triggers the DNA damage response and activates the apoptotic pathway, by down-regulating autophagy and DNA repair system in HCT-116 cells. TAN and 5-FU co-treatment also remarkably reduces the mitochondrial membrane potential, and sequentially decreases ATPase activity. Collectively, results indicate that combination of TAN and 5-FU significantly accelerates apoptosis via JNK mediated pathway. To our knowledge gained from literature, this study is the first to describe synergistic activity of TAN and 5-FU against colorectal cancer cells.Copyright © 2020 Elsevier Ltd. All rights reserved.