Introduction: Although the survival rate of colorectal cancer (CRC) patients can be improved by surgery, radiotherapy, and chemotherapy, the resistance to 5-fluorouracil (5-Fu) affects the effect of chemotherapy and the prognosis of patients. An increasing number of studies showed that 5-Fu resistance was the main reason for the failure of colorectal cancer treatment. The poor prognosis of colorectal cancer greatly harms people’s health. This study aimed to clarify the correlation between cyclin-dependent kinase 1 (CDK1) and 5-Fu-induced tumor resistance. Materials and Methods: Cell proliferation and invasion experiments showed that down-regulation of CDK1 inhibited fluorouracil-resistant CRC cell proliferation. The expression level of CDK1 was detected in 5-Fu-resistant CRC cells in vitro. Tumor growth was inhibited by down-regulation of CDK1 in tumor xenograft mouse models. Results: We found that CDK1 was highly expressed in tumor tissues, especially in fluorouracil-resistant tissues. We also confirmed that the differential expression of 5-Fu in tumor tissues was related to tumor site, lymph node metastasis and stage. CDK1 promoted migration, invasion and inhibited apoptosis in 5-Fu-resistant CRC cells. Down-regulation of CDK1 inhibited fluorouracil-resistant CRC cell proliferation and tumorigenesis in vivo. Conclusion: High expression of CDK1 may lead to poor clinical prognosis, and inhibition of CDK1 enhances 5-Fu sensitivity in CRC. Our research suggested that CDK1 may be used to predict 5-Fu efficacy and as a therapeutic target for CRC.