Radiotherapy is a key component of the standard treatment for esophageal squamous cell carcinoma (ESCC); however, therapeutic efficacy is frequently limited by resistance. Elucidating the mechanisms governing radiosensitivity may facilitate the development of improved therapeutic strategies for patients with ESCC. In this study, we demonstrated that ANXA2 knockdown (KD) inhibited the proliferation, migration, and invasion of ESCC cells and promoted their radiosensitivity. Mechanistically, we demonstrated that ANXA2 KD in ESCC cell lines inhibited protective autophagy, whereas the activation of autophagy by treatment with Earle’s balanced salt solution (EBSS) reversed this effect. ANXA2 expression was downregulated and upregulated by the overexpression and knockdown of tripartite motif-containing protein 21 (TRIM21), respectively. TRIM21 regulated ANXA2 stability through the ubiquitination process, thereby affecting autophagic activity in ESCC cells and ultimately affecting ESCC progression and radiosensitivity. Our findings suggest that the TRIM21/ANXA2/Beclin1 (autophagy-related protein 6) axis regulates ESCC cell radiosensitivity through the induction of autophagy, indicating that targeting this signaling axis may provide new insights into ESCC radiosensitivity.