Iodine-125 (125 I) seed brachytherapy has been proven to be a safe and effective treatment for advanced esophageal cancer; however, the mechanisms underlying its actions are not completely understood. In the present study, the anti-cancer mechanisms of 125 I seed radiation in human esophageal squamous cell carcinoma (ESCC) cells (Eca-109 and KYSE-150) were determined, with a particular focus on the mode of cell death. The results showed that 125 I seed radiation significantly inhibited cell proliferation, and induced DNA damage and G2/M cell cycle arrest in both ESCC cell lines. 125 I seed radiation induced cell death through both apoptosis and paraptosis. Eca-109 cells were primarily killed by inducing caspase-dependent apoptosis, with 6 Gy radiation resulting in the largest response. KYSE-150 cells were primarily killed by inducing paraptosis, which is characterized by extensive cytoplasmic vacuolation. 125 I seed radiation induced autophagic flux in both ESCC cell lines, and autophagy inhibition by 3-methyladenine enhanced radiosensitivity. Furthermore 125 I seed radiation induced increased production of reactive oxygen species (ROS) in both ESCC cell lines. Treatment with an ROS scavenger significantly attenuated the effects of 125 I seed radiation on endoplasmic reticulum stress, autophagy, apoptosis, paraptotic vacuoles and reduced cell viability. In vivo experiments showed that 125 I seed brachytherapy induced ROS generation, initiated cell apoptosis and potential paraptosis, and inhibited cell proliferation and tumor growth. In summary, the results demonstrate that in ESCC cells, 125 I seed radiation induces cell death through both apoptosis and paraptosis; and at the same time initiates protective autophagy. Additionally, 125 I seed radiation-induced apoptosis, paraptosis and autophagy was considerably mediated by ROS.