Autophagy has been reported to play a radioresistance role in high-dose-rate irradiation. However, its mechanisms and roles in continuous low-dose-rate (CLDR) irradiation have not been clearly understood. Iodine-125 (I-125) seed brachytherapy is a modality of CLDR irradiation and has been used in the treatment of various cancers. In this study, we investigated the mechanisms and roles of autophagy induced by I-125 seed radiation in human esophageal squamous cell carcinoma (ESCC) cell lines (Eca-109 and EC-109) and a xenograft mouse model. The results of this work showed that I-125 seed radiation induced a dose-dependent increase in autophagy in both cell lines. In Eca-109 cells, I-125 seed radiation-induced endoplasmic reticulum (ER) stress, manifesting as the increased levels of intracellular Ca²⁺ and Grp78/BiP, and activated PERK-eIF2α, IRE1, and ATF6 pathways of the unfolded protein response. Knockdown of PERK led to the decreased expression of autophagy marker, LC3B-II. Inhibition of autophagy by chloroquine or knockdown of ATG5 enhanced I-125 seed radiation-induced cell proliferation inhibition and apoptosis. Interestingly, chloroquine did not aggravate ER stress but promoted apoptosis via the mitochondrial pathway. The animal experiment showed that inhibition of autophagy by chloroquine improved the efficacy of I-125 seed radiation. In summary, our data demonstrate that I-125 seed CLDR radiation induces ER stress-mediated autophagy in ESCC. Autophagy plays a pro-survival role in I-125 seed CLDR irradiation, and chloroquine is a potential candidate for use in combination therapy with I-125 seed radiation treatment to improve efficacy against ESCC.