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Esophageal squamous cell carcinoma (ESCC) has high aggressiveness and poor prognosis, and is the major histological subtype of esophageal cancer in East Asia and East Africa. In this study, we found that USP46, a deubiquitinating enzyme, is overexpressed in clinical ESCC samples, especially in patients with positive lymph node metastasis. Moreover, USP46 enhances the migration and invasion of ESCC cells by mediating the EMT process in vitro , and promotes lymph nodes and lung metastasis of ESCC in vivo. In addition, we found that USP46 is a bona fide deubiquitinating enzyme to stabilize the protein level of ENO1 through deubiquitination. ENO1 protein level was also positively correlated with USP46 in the ESCC samples. In summary, these findings reveal the functional role of USP46 as a deubiquitinating enzyme on ESCC metastasis, providing us a potential therapeutic target for the treatment of ESCC. • USP46 is highly expressed in esophageal squamous cell carcinoma (ESCC). • USP46 promotes tumor metastasis of ESCC in vitro and in vivo. • USP46 promotes EMT process in ESCC. • USP46 is a bona fide deubiquitinating enzyme to regulate ENO1 stability. • USP46 and ENO1 expression are uniformly correlated with lymph node metastasis in ESCC. [ABSTRACT FROM AUTHOR]