Abstract: Objective To investigate the effect and mechanism of Xuanfu Daizhe Decoction on the proliferation, migration, and invasion activities of esophageal cancer cells. Methods Human esophageal cancer cell line EC109 was treated with Xuanfu Daizhe Decoction at different concentrations, and the cells were divided into high-concentration group (200 μg/mL), medium-concentration group (100 μg/mL), low-concentration group (50 μg/mL), and blank group (0 μg/mL) based on the concentration. CCK-8 assay, wound healing assay, and Transwell invasion assay were used to detect the proliferation, migration, and invasion activities of the cells in each group, respectively. Western blot and cellular immunofluorescence staining were employed to detect the protein expression levels of glycolysis-related enzymes hexokinase 2 (HK2), lactate dehydrogenase A (LDHA), and phosphofructokinase 1 (PFK1) in the cells of each group, and real-time quantitative fluorescent polymerase chain reaction (qRT-PCR) was used to detect the relative expression levels of mRNA encoding these enzymes. A nude mouse tumor-bearing model was established and divided into control group and Xuanfu Daizhe Decoction-fed group (20 mg/kg) to observe the effect of Xuanfu Daizhe Decoction on the growth of esophageal cancer in vivo. Results The results of CCK-8 assay, wound healing assay, and Transwell invasion assay showed that Xuanfu Daizhe Decoction could inhibit the proliferation, migration, and invasion activities of EC109 cells in a concentration-dependent manner. Western blot and cellular immunofluorescence analysis revealed that compared with the blank group, the protein expression levels of HK2, LDHA, and PFK1 in the low-concentration, medium-concentration, and high-concentration groups were decreased (P<0.05). The qRT-PCR results showed that compared with the blank group, the relative expression levels of HK2 mRNA, LDHA mRNA, and PFK1 mRNA in the low-concentration, medium-concentration, and high-concentration groups were all reduced(P<0.01 or P<0.000 1). The tumor volume in the subcutaneous tissue on the back of nude mice in the Xuanfu Daizhe Decoction-fed group was smaller than that in the control group, and the protein expression level of LDHA in the tumor tissue of the Xuanfu Daizhe Decoction-fed group was lower than that in the control group, while the expression level of the pro-apoptotic protein Bax was higher than that in the control group (P<0.05). Conclusion Xuanfu Daizhe Decoction can inhibit the glycolysis process of esophageal cancer cells in a concentration-dependent manner, thereby inhibiting cell proliferation, migration, invasion, and tumor growth in vivo.