Objective To explore the molecular mechanism of ethyl acetate extract of Liujunzi Decoction (EAELD) on energy metabolism in esophageal cancer EC9706 cells in conditioned medium from cancer-associated fibroblasts (CAFs). Methods Methyl thiazol tetrazolium assays were used to assess the effect of EAELD on EC9706 cell proliferation. The effects of EAELD on lactate and glucose in the culture supernatant of EC9706 cells in CAF-conditioned medium were assessed by colorimetry. A seahorse system for energy metabolism analysis was used to assess the effect of EAELD on energy metabolism of EC9706 cells in CAF-conditioned medium. Real-time quantitative PCR (qPCR) and Western blot were used to measure mRNA and protein expression of energy metabolism-related molecules. Results Compared with DMEM, except for the 10 μg/ mL group, EAELD had a significant inhibitory effect on EC9706 cell proliferation (P<0. 05). The 30% inhibitory concentration (IC₃₀) of 25 μg/ mL and half inhibitory concentration (IC₅₀) of 40 μg/ mL were selected as low and high doses for subsequent experiments. In EC9706 cells cultured in CAFM, both low and high dose EAELD groups had significantly reduced non-mitochondrial oxygen consumption, basal respiration, maximum respiration, oxygen consumption of ATP synthesis, spare respiration capacity, basal glycolysis, compensative glycolysis, and glycolysis potential (P<0. 01). Lactate content of EC9706 cells was decreased (P<0. 01), mRNA expression of GLUT1 (P< 0. 05, P< 0. 01) was downregulated, and protein expression of p-PKM2, HK2, PKM2 and MCT1 was downregulated (P<0. 01). The high dose EAELD group had downregulated mitochondrial oxygen consumption and basal respiration. The glycolytic ratio of (P<0. 05) and glucose uptake of EC9706 cells were reduced (P<0. 05) and protein expression of p-PKM2 and GLUT1 was downregulated ( P< 0. 01, P< 0. 05). The low dose group of EAELD had downregulated mRNA expression of MCT1 (P<0. 05). Conclusions EAELD interferes with the energy metabolism of EC9706 cells in CAF-conditioned medium, and its mechanism may be related to regulation of HK2, PKM2, GLUT1, MCT1 and MCT4 mRNA and protein expression.