Objective: To explore the molecular mechanism of ethyl acetate extract of Liujunzi Decoction(EAELD) on energy metabolism of esophageal cancer EC9706 cells in conditioned medium of cancer-associated fibroblasts (CAFs). Methods: Methyl thiazol tetrazolium (MTT) assay was used to detect the effect of EAELD on the proliferation activity of EC9706. The effects of EAELD on lactate and glucose in the supernatant of EC9706 cells in CAFs conditioned medium were detected by colorimetry. seahorse system energy metabolism analysis system was used to detect the effect of EAELD on energy metabolism of EC9706 cells in CAFs conditioned medium. Real-time fluorescent quantitative PCR (q-PCR) and western blotting were used to detect the mRNA and protein expression of energy metabolism-related molecules.. Results: Compared with DMEM, except for the 10μg/mL group,EAELD had a significant inhibitory effect on the proliferation of EC9706 cells (P < 0.05). The inhibitory concentration (IC30) of 25μg/mL and half inhibitory concentration (IC50) of 40μg/mL were selected as the low and high dose groups for subsequent experiments. Among EC9706 cells cultured by CAFM, both low-dose and high-dose EAELD groups could significantly reduce Non-mitochondrial oxygen consumption, Basal respiration value, Maximum respiration value, Oxygen consumption of ATP synthesis, Spare respiration capacity, Basal glycolysis, Compensative glycolysis and glycolysis potential (P <0.01). Decreased the lactate content of EC9706 cells (P <0.01), down-regulated the mRNA expression of GLUT1 (P <0.05, P <0.01), down-regulated the protein expression of p-PKM2, HK2, PKM2 and MCT1 (P <0.01); The high-dose EAELD group could down-regulate the Mitochondrial oxygen consumption and basal use The glycolytic ratio of EC9706 cells (P <0.05), reduce glucose uptake of EC9706 cells (P <0.05), down-regulate the protein expression of p-PKM2 and GLUT1 (P <0.01, P <0.05); The low dose group of EAELD could down-regulate the mRNA expression of MCT1 (P <0.05). Conclusions:EAELD can interfere with the energy metabolism of EC9706 cells in CAFs conditioned medium, and its mechanism may be related to the regulation of HK2, PKM2, GLUT1, MCT1 and MCT4 mRNA and protein expression.