Cholangiocarcinoma (CCA) is the second most common primary liver malignancy, however, it is difficult to diagnose and treat, and only a few patients with CCA are suitable for surgery. Iodine-125 (I-125) is an effective treatment for cancer, but the molecular mechanisms underlying the effects of I-125 differ among different cancers. This study aimed to explore the effects of I-125 on CCA cell activity and determine the possible mechanisms of action of I-125 in this type of cancer. CCA cell proliferation, cycling, apoptosis, autophagy, and endoplasmic reticulum (ER) stress were determined after irradiation of CCA cells with I-125 seeds. The effects of I-125 on autophagy and ER stress in three CCA cell lines were evaluated using western blotting, while the effects of I-125 on apoptosis and autophagy in QBC939 cells treated with si-Beclin1 or si-PERK, respectively, were assessed using flow cytometry. I-125 suppressed cell viability and induced cell cycle G2/M-phase arrest in three CCA cell lines (QBC939, TFK-1, HuCCT1). I-125 induced apoptosis, autophagy, and ER stress by altering the expression levels of some related proteins in each of the three CCA cell lines. Furthermore, autophagy inhibition (treatment with si-Beclin1) increased expression of apoptosis-related proteins (cleaved-PARP and cleaved-caspase-3, Bax/Bcl2) in QBC939 cells irradiated with I-125 seeds, while ER stress inhibition (with si-PERK) suppressed the expression of autophagy-related proteins (LC3-I, LC3-II, p62). Therefore, I-125 induces ER stress, thereby activating protective autophagy in CCA cells through the PERK signaling pathway. Combined inhibition of ER stress and autophagy signaling may increase the killing effect of I-125 on cancer cells and serve as a new auxiliary method in I-125 radiotherapy.