Abstract: This study aims to compare the killing effect of carbon ions and X-rays on renal cell carcinoma 786-O cells and to explore the differential mechanism from the perspective of endoplasmic reticulum stress and autophagy regulation. Colony formation, CCK-8, and scratch wound healing assays were used to detect cell proliferation, survival, and migration ability. Transmission electron microscopy was used to observe changes in the endoplasmic reticulum ultrastructure. Western blot was used to detect the expression of the endoplasmic reticulum stress marker proteins GRP78 and CHOP, as well as the autophagy-related protein LC3Ⅱ. qRT-PCR was used to detect the mRNA level of the autophagy-related gene LC3B. The autophagy inhibitors 3-methyladenine (3-MA) and chloroquine (CQ) were combined with X-ray treatment to evaluate the effect on cellular radiosensitivity. The results showed that after carbon ion irradiation, the colony formation ability, proliferation activity, and migration ability of the cells were significantly lower than those in the X-ray group. Carbon ion irradiation caused severe swelling and fragmentation of the endoplasmic reticulum, while X-ray irradiation only caused mild damage. Although carbon ions induced more severe structural damage to the endoplasmic reticulum, the upregulation of GRP78 and CHOP was lower than that in the X-ray group. X-rays significantly upregulated LC3B/LC3Ⅱ expression. 3-MA combined with X-rays decreased LC3B/LC3Ⅱ expression, whereas CQ combined with X-rays led to LC3B/LC3Ⅱ accumulation; both autophagy inhibitors further reduced the cell survival rate after X-ray irradiation. These results indicate that the stronger killing effect of carbon ions on renal cell carcinoma cells is related to the irreversible structural damage it induces to the endoplasmic reticulum, which impairs the cells’ ability to initiate protective endoplasmic reticulum stress and autophagy. In contrast, the protective autophagy induced by X-rays reduces its killing effect. This study reveals, for the first time, the radiobiological mechanism by which heavy ions are superior to photons from the perspective of endoplasmic reticulum stress and autophagy regulation, providing possible new targets for the clinical application of heavy ion radiotherapy and for radiosensitization strategies in photon radiotherapy.