<p>Synergistic therapy is unwaveringly developing as a potential approach to enhance the effectiveness of anticancer medications in chemotherapy. Incorporating adjuvants alongside conventional anticancer medications has demonstrated selective cytotoxicity toward cancer cells while mitigating adverse effects such as DNA damage and systemic toxicity in normal cells. The extended use of 5-FU, a synthetic medicine used in chemotherapy, has many adverse effects, which limit its long-term use in clinical settings. Numerous investigations have demonstrated that luteolin exhibits anticancer effects and alleviates inflammation in mammalian cells. In this study, we provide the first experimental evidence of the synergistic interaction between luteolin and 5-FU in murine melanoma (B16F10) cells, compared to monotherapies (luteolin and 5-FU alone). Using MTT assay for cell viability, flow cytometry for cell cycle analysis, and Western blotting for apoptosis markers, we demonstrate that simultaneous exposure to luteolin and 5-FU speeds up oxidative stress and enhances the production of endogenous ROS. The combination therapy induced the G1-phase cell cycle arrest (65.39%) and decreased the cell viability of B16F10 cells to 34.6%. This, in turn, activates the DNA damage response and initiates the apoptotic pathway by suppressing autophagy and the DNA repair system. Further, the co-treatment of luteolin and 5-FU significantly decreases the mitochondrial membrane potential and reduces ATP production. The results indicate that the combination of luteolin and 5-FU may improve treatment efficacy while potentially decreasing the necessary dosage of 5-FU, therefore reducing its toxicity in clinical settings.</p>