Objective This study aimed to investigate the synergistic effects of low-dose aspirin and caffeine for preventing colorectal cancer. Methods The colorectal cell lines Caco-2,SW-480,HT-29,and HCT-116 were exposed to low-dose aspirin alone,caffeine alone or both agents,then cell viability was measured using the cell counting kit-8 and extent of apoptosis was measured using flow cytometry. Expression of relevant factors(p53,PTEN,EGFR,TNF-α) was measured using real-time PCR. Drug-withdrawal experiments were conducted. Results Treating cell lines with both caffeine and aspirin inhibited cell proliferation and accelerated early apoptosis significantly more than treating cells with either agent alone. Western blotting showed that treating with aspirin alone caffeine alone and with the combination of aspirin and caffeine up-regulated p53,Cyt-C and caspase-9/-3 expression, and the difference of treatment with the combination of aspirin and caffeine was the most significant(P<0.05). The level of cell vitality were improved,when the p53-specific inhibitor pifithrin-α were added. In drug-withdrawal experiments, caffeine treatment prolonged p53 up-regulation and inhibition of proliferation. Conclusion Aspirin and caffeine synergistically inhibit proliferation and promote apoptosis of colorectal cancer cells. In addition, maintaining caffeine can prolong growth inhibition after aspirin withdrawal.