background: Since tumors need oxygen and nutrients to grow and spread, angiogenesis has an essential role in cancer growth and metastasis. So, inhibition of angiogenesis at the initial stage of cancer is a critical strategy in metastasis inhibition. 5-Fluorouracil (5-FU) is a chemotherapy drug that has also been shown to have anti-angiogenic effects. Quercetin, a natural polyphenolic compound, has anti-angiogenic effects. The present study was conducted to investigate the enhancement of the anti-angiogenic effect of 5-FU in combination with Quercetin (Que) and compare it with the application of 5-FU alone.Method and Results: Following the treatment of human umbilical vein endothelial cells (HUVECs) with Que, 5-FU and their combination, the cell viability, migration, and gene expression of VEGFR2 and VEGFR1 were assessed through MTT assay, wound healing assay, and real-time RT-PCR, respectively. In vivo angiogenesis was evaluated using chicken chorioallantoic membrane (CAM) assay. Our study showed that cell viability, migration, gene expression of VEGFR2 and VEGFR1, and angiogenesis significantly decreased following Que and 5-FU alone treatment, and the decrease in combination state was significant compare to 5-FU alone.Conclusions: In summary, the present study showed that the combination of Que with 5-FU improves its anti-angiogenic effects. Therefore, this combination can be suggested for future in vivo studies.