Stress granules (SGs) are formed through liquid-liquid phase separation (LLPS), in response to external stimuli. YBX1, an integral component of SGs, plays a crucial role in tumor progression and cellular stress response. This study aims to elucidate the mechanisms and specific biological implications of YBX1 in SG formation, along with the identification of key regions and interacting proteins. Our observations indicate that YBX1 rapidly undergoes liquid-liquid phase separation, leading to SG formation in response to 8 Gy X-ray irradiation within 1 h, with SGs reverting to their original state after 5 h. There was a potential interaction between ATXN2L and YBX1, persisting YBX1 within the SGs. Our data suggested a potential interaction between ATXN2L and YBX1, and it remained associated with YBX1 within the SGs. Furthermore, our subsequent studies demonstrate that targeting ATXN2L can diminish the recruitment of YBX1 to stress granules (SGs), consequently enhancing the radiosensitivity of HeLa cells.