Background <p>Ubiquitin-specific proteases (USPs) play a critical role in the development of various cancers. The study aimed to elucidated the pathogenic molecular mechanisms and analyze its clinical significance in esophageal squamous cell carcinoma (ESCC).</p> Methods <p>Pyrosequencing, RT-qPCR and Western blot were used to detect the promoter DNA methylation level and expression of USP44 in ESCC tissues and cell lines, and the significance of USP44/SENP2 in the progression of ESCC was evaluated by in vitro and in vivo experiments. The regulatory mechanism of USP44 on SENP2 was explored by liquid chromatography-mass spectrometry, co-immunoprecipitation (Co-IP) combined with cycloheximide (CHX) tracking assay and ubiquitination analysis. Finally, 182 ESCC tissues were stained with immunohistochemistry (IHC), and the correlation between USP44 and prognosis was evaluated by Kaplan–Meier survival analysis, Cox regression and Nomogram model.</p> Results <p>In ESCC tumor tissues and cell lines, USP44 is frequently hypermethylated and downregulated. Overexpression of USP44 significantly inhibits ESCC cell invasion and metastasis both in vitro and in vivo. Mechanistically, USP44 interacts with and stabilizes ubiquitin-like modifier (SUMO)-specific peptidase 2 (SENP2) through deubiquitination, thereby inhibiting the progression of ESCC. Knockdown of SENP2 reduced the inhibitory effect of USP44 on ESCC cell migration and invasion. Clinically, low expression of USP44 indicates poor prognosis and is an independent prognostic factor in ESCC patients, as evidenced by Kaplan–Meier curve and cox regression analysis (HR = 0.345, 95% CI 0.227-0.524, <i>p</i> &lt; 0.001). We established a nomogram model with robust predictive power for the 1-, 3-, and 5-year survival of ESCC patients (AUC = 0.849).</p> Conclusion <p>USP44-SENP2 axis is a pivotal factor in the progression of ESCC, and provides potential therapeutic targets for ESCC patients.</p>