Inducing cell senescence is widely regarded as a potent tumor suppression mechanism. Resveratrol has attracted increasing attention for its capacity to prevent and suppress cancer. However, the mechanism of resveratrol on the induction of cancer cell senescence has not been well clarified. Our results showed that resveratrol inhibited cell viability and colony formation and promoted cell senescence along with augmentation of SA-β-gal activity and modulation of senescence-associated molecular markers p53, p21 and LaminB protein in breast and liver cancer cells. The underlying mechanism was that resveratrol increased ROS generation to enhance tumor suppressor gene DLC1 expression, and DLC1 further inhibited the DYRK1A-EGFR axis to trigger DNA damage accompanied by up-regulation of the DNA double strand break marker protein γH2AX and down-regulation of the DNA repair related proteins p-BRCA1 and RAD51, eventually leading to cancer cell senescence. Resveratrol also effectively inhibited the volume of transplanted tumor with increased SA-β-gal activity and DLC1 level in a chicken embryo allantoic membrane xenograft tumor model. This is the first report to investigate whether resveratrol induces DNA damage-mediated cancer cell senescence through the DLC1-DYRK1A-EGFR axis, which could provide a solid base for resveratrol's application in cancer prevention and clinical treatment as a food additive or adjuvant therapies.