This study investigates the role of Serine Hydroxymethyl Transferase 2 (SHMT2) in esophageal cancer (EC) under hypoxic conditions. SHMT2 is a key metabolic enzyme frequently expressed in human malignancies. The research team assessed SHMT2 levels in EC tissues and cell lines (TE-1 and EC109) using RT-qPCR. They found that SHMT2 expression was significantly upregulated in EC tissues and cells, and hypoxia further increased SHMT2 protein expression. Hypoxia enhanced EC cell proliferation, migration, invasion, stemness, and glycolysis. Additionally, hypoxia triggered lactylation of the SHMT2 protein, increasing its stability. SHMT2 knockdown impeded the malignant phenotype of EC cells. Mechanistically, SHMT2 interacts with MTHFD1L, and hypoxia-induced SHMT2 lactylation upregulates MTHFD1L expression, accelerating EC progression. The study highlights the importance of SHMT2 in the malignant behavior of EC cells under hypoxic conditions and suggests potential therapeutic targets for EC treatment.This study investigates the role of Serine Hydroxymethyl Transferase 2 (SHMT2) in esophageal cancer (EC) under hypoxic conditions. SHMT2 is a key metabolic enzyme frequently expressed in human malignancies. The research team assessed SHMT2 levels in EC tissues and cell lines (TE-1 and EC109) using RT-qPCR. They found that SHMT2 expression was significantly upregulated in EC tissues and cells, and hypoxia further increased SHMT2 protein expression. Hypoxia enhanced EC cell proliferation, migration, invasion, stemness, and glycolysis. Additionally, hypoxia triggered lactylation of the SHMT2 protein, increasing its stability. SHMT2 knockdown impeded the malignant phenotype of EC cells. Mechanistically, SHMT2 interacts with MTHFD1L, and hypoxia-induced SHMT2 lactylation upregulates MTHFD1L expression, accelerating EC progression. The study highlights the importance of SHMT2 in the malignant behavior of EC cells under hypoxic conditions and suggests potential therapeutic targets for EC treatment.