5‑Fluorouracil (5‑FU) is a commonly used anti‑tumor chemotherapeutic drug for cervical carcinoma. However, increased drug resistance may occur following several cycles of 5‑FU‑based chemotherapy. Novel strategies of gene therapy for enhancing the sensitivity of cancer cells to 5‑FU chemotherapy have been intensively explored. Human telomerase reverse transcriptase (hTERT)C27 is a newly constructed hTERT C‑terminal polypeptide that is capable of promoting chromosome end‑to‑end fusion during anaphase and inducing telomere dysfunction. In the present study, the effects of hTERTC27 overexpression on 5‑FU‑induced proliferation inhibition and apoptosis were observed. HeLa cells were cultured and transfected with the constructed pcDNA3.1 or pcDNA3.1‑hTERTC27 vectors. Expression of hTERTC27 was detected using western blot analysis and was assessed using MTT assays and flow cytometry. The results demonstrated that overexpressed hTERTC27 increased the sensitivity of the HeLa cells to 5‑FU and significantly inhibited HeLa cell proliferation with 5‑FU treatment. In addition, hTERTC27 overexpression evidently promoted the 5‑FU‑induced apoptosis by increasing the expression of activated caspase‑3 and ‑9 and by downregulating the expression of B‑cell lymphoma 2. The results suggest that hTERTC27 overexpression is a potential clinical strategy for enhancing the antitumor effect of 5‑FU chemotherapy in the treatment of cervical carcinoma.