Esophageal carcinoma is the tenth most common cancer in the world. long non-coding RNA (lncRNA) is a type of RNA molecule that has no or limited coding ability. Many studies have found that it plays an important role in the occurrence and development of tumors. This article aims to investigate the expression and clinical significance of lncRNA VPS9D1-AS1 in patients with esophageal squamous cell carcinoma (ESCC). Additionally, it aims to examine the effects of targeted silencing of VPS9D1-AS1 on the growth and migration of ESCC cells in vivo and in vitro, and to explore the mechanism of action. This study compared the expression difference of lncRNA VPS9D1-AS1 in ESCC tissues and normal tissues through TCGA and UCSC databases, and analyzed the correlation between VPS9D1-AS1 expression and the prognosis of ESCC patients; clinical samples from ESCC patients were collected and qRT-PCR (Fluorescent quantitative polymerase chain reaction) was used to detect the relative expression of VPS9D1-AS1 and analyze the correlation between the expression of VPS9D1-AS1 and the onset and development of ESCC. Specific small interfering RNA was used to knock down the expression of VPS9D1-AS1 in ESCC cells, and the effect of VPS9D1-AS1 on the proliferation and migration of esophageal squamous cell carcinoma cells was detected by CCK-8 and Transwell assays. A subcutaneous transplantation tumor model of ESCC cells was established, and cholesterol-modified si-VPS9D1-AS1 was injected around the tumor to detect the impact of VPS9D1-AS1 on ESCC tumor growth in vivo. Western blot was used to detect the expression of apoptosis-related proteins in the tumor tissues of mice in each group. Finally, it was found that lncRNA VPS9D1-AS1 was highly expressed in ESCC tissues and significantly correlated with the prognosis of ESCC patients. Knocking down VPS9D1-AS1 regulates the proliferation, migration, and apoptosis of ESCC cells, and inhibits the growth of transplanted tumors in vivo, indicating it is a potential diagnostic and therapeutic target for esophageal squamous cell carcinoma.