Hypoxia-induced SHMT2 protein lactylation promotes glycolysis and stemness in esophageal cancer (EC) cells. Esophageal cancer is a highly lethal digestive tract tumor, with hypoxia playing a critical role in tumor progression and abnormal energy metabolism. SHMT2, a key metabolic enzyme, is significantly upregulated in EC tissues and cells under hypoxia, enhancing cell proliferation, migration, invasion, stemness, and glycolysis. Hypoxia increases SHMT2 protein expression and stability, leading to its lactylation, which further enhances its expression and promotes malignant progression. Mechanistic studies reveal that SHMT2 interacts with MTHFD1L, and hypoxia-induced lactylation of SHMT2 increases MTHFD1L expression, accelerating EC progression. Lactylation, a novel post-translational modification, regulates cellular metabolism and function by modulating protein stability and activity. SHMT2 is essential for tumor cell survival under hypoxia, and its lactylation under hypoxia may be a novel therapeutic target for EC. The study highlights the importance of understanding the molecular mechanisms underlying hypoxia-induced metabolic changes in EC, which could lead to the development of new therapeutic strategies. The findings suggest that targeting SHMT2 lactylation may be a promising approach for improving the prognosis of EC patients.Hypoxia-induced SHMT2 protein lactylation promotes glycolysis and stemness in esophageal cancer (EC) cells. Esophageal cancer is a highly lethal digestive tract tumor, with hypoxia playing a critical role in tumor progression and abnormal energy metabolism. SHMT2, a key metabolic enzyme, is significantly upregulated in EC tissues and cells under hypoxia, enhancing cell proliferation, migration, invasion, stemness, and glycolysis. Hypoxia increases SHMT2 protein expression and stability, leading to its lactylation, which further enhances its expression and promotes malignant progression. Mechanistic studies reveal that SHMT2 interacts with MTHFD1L, and hypoxia-induced lactylation of SHMT2 increases MTHFD1L expression, accelerating EC progression. Lactylation, a novel post-translational modification, regulates cellular metabolism and function by modulating protein stability and activity. SHMT2 is essential for tumor cell survival under hypoxia, and its lactylation under hypoxia may be a novel therapeutic target for EC. The study highlights the importance of understanding the molecular mechanisms underlying hypoxia-induced metabolic changes in EC, which could lead to the development of new therapeutic strategies. The findings suggest that targeting SHMT2 lactylation may be a promising approach for improving the prognosis of EC patients.