Abstract: Objective To explore the effects and mechanism of Pin2/TRF1 interacting protein X1 (PinX1) gene on the prognosis and 5-fluorouracil (5-FU) sensitivity of colorectal cancer (CRC). Methods The expression and prognosis of PinX1 in CRC tissues were analyzed by online GEPIA2 database. A plasmid over-expression pEGFP-C3-PinX1 was constructed and transfected into CRC cell lines (LoVo and HT29). CCK-8 assay was used to determine cell proliferation. 5-FU dose-response curve was plotted to calculate IC₅₀. Real-time quantitative PCR was performed to detect relative telomere length (RTL). Apoptosis was detected by flow cytometry. Results High expression of PinX1 was associated with early CRC staging and better overall survival (OS). According to Western blot, PinX1 was successfully transfected. In the PinX1 overexpression groups, LoVo and HT29 cells showed a declined proliferation rate, with decreases in the IC₅₀ value of 5-FU and RTL, and increases in apoptotic rate, compared to the empty vector group. The changes in RTL and apoptosis rate became more significantly after combined treatment with a low dose of 5-FU (0.5 mg/L). Conclusions Up-regulation of PinX1 mRNA indicates better prognosis of CRC pateints. PinX1 overexpression can inhibit the proliferation of CRC cells and increase the sensitivity of 5-FU, which may be related to shortened telomere length and induced apoptosis.