Homeobox containing 1 (HMBOX1) modulates telomere length in various types of tumor cells by binding to double‑stranded telomeric DNA. There is a negative correlation between telomere length and radiosensitivity in tumor cells. In the present study, we aimed to investigate the relationship among HMBOX1, telomere and radiosensitivity in cervical cancer cells. Lentivirus-based shRNAs were used to establish stable transfected cell lines in which protein and mRNA levels of HMBOX1 were notably decreased. Knockdown of HMBOX1 increased the radiosensitivity of HeLa and C33A cells. TERT protein was also decreased while HMBOX1 was downregulated. Knockdown of HMBOX1 shortened telomere length in the HeLa cells, while TERT overexpression rescued telomere shortening in the HeLa-HMBOX1 cells. Knockdown of HMBOX1 increased the apoptosis rate, decreased radiation-induced DNA damage foci, and inhibited the expression of ATM, ATR, p-ATM, p-ATR and BRCA1 in the homologous recombination repair pathway. Our data suggest a possible role of HMBOX1 in regulating radiosensitivity in cervical cancer cells. Moreover, HMBOX1 may be a potential factor in the radiotherapy of cervical cancer.