Acquired radioresistance is a major clinical obstacle in the treatment of esophageal cancer (EC). Ubiquitin-specific protease 28 (USP28) has been implicated in tumor growth in various cancer types. However, the role of USP28 and its underlying mechanisms of radioresistance in EC remain unknown. In the current study, we found that USP28 and c-Myc levels were upregulated in EC tissues and EC cell lines. The mRNA expression levels of USP28 and c-Myc were increased in the radioresistant human EC cell line (ECA109R) compared with those in ECA109 cells. In addition, the expression levels of USP28 and c-Myc were increased with increase in culture time after irradiation. Meanwhile, overexpression of USP28 decreased the radiosensitivity of ECA109 cells. In contrast, USP28 knockdown enhanced the radiosensitivity of ECA109R cells. Moreover, USP28 positively regulated the protein level of c-Myc, and c-Myc negatively regulated the radiosensitivity of ECA109 and ECA109R cells. Furthermore, c-Myc reversed the inhibitory effect of USP28 on the radiosensitivity of EC cells. Additionally, c-Myc enhanced the accumulation of hypoxia-inducible factor-1 alpha (HIF-1α) at the posttranscriptional level, and the reinforcing effect of c-Myc silencing on the radiosensitivity of EC cells could be reversed by HIF-1α overexpression. Besides, knockdown of USP28 blocked the effect of c-Myc on activation of ataxia telangiectasia-mutated/ataxia telangiectasia and Rad3-related DNA damage checkpoint after irradiation. In conclusion, knockdown of USP28 enhanced the radiosensitivity of EC cells by destabilizing c-Myc and enhancing the accumulation of HIF-1α. Therefore, USP28 may serve as a novel therapeutic target to overcome EC radioresistance.