Abstract Introduction: Esophageal carcinoma (EC) ranks eighth in the incidence of malignant tumors and the sixth mortality worldwide. This study aims to screen out the Chinese herbs with strong inhibitory effects on the proliferation of four kinds of EC cells EC9706, EC-1, TE-1 and Eca109 from 366 Chinese herbs produced in Henan Province, and study the effect of composed Removing Heat and Nourshing Yin Prescription(RHNYP) on the proliferation, migration, invasion and PI3K/AKT signaling pathway of four EC cells. Methods: MTT and orthogonal design was applied to screen out the best effective Chinese herbs and determine the optimal dosage ratio of prescription on EC9706, EC-1, TE-1, Eca109 cells from 366 Chinese herbs alcohol extracts, the effects of this prescription on the proliferation, migration, invasion, clone formation, cell cycle and apoptosis of four EC cells were investigated by MTT, RTCA, soft agarose colony forming test, flow cytometry and western blot analysis. Then exploring the effects of RHNYP on EC related signaling pathways and key genes expression by western blot and RT-PCR assay. Results: Rhizoma Fagopyri Dibotryis(Jinqiaomai), Rhizoma Anemarrhenae(Zhimu), Asparagus Cochinchinensis(Tiandong), and Carpesium Abrolanoides Linne(Heshi) were screened out and composed Removing Heat and Nourshing Yin Prescription(RHNYP) according to their efficacy and orthogonal design. RHNYP could obviously inhibit the proliferation of EC9706, TE-1, EC-1, ECa109 cells with dose-effect and time-dependent manner. IC50 values of Jinqiaomai, Zhimu, Tiandong, Heshi in RHNYP for EC9706 cell were 27.639μg/ml, 40.015μg/ml, 9.044μg/ml, 11.208μg/ml respectively; for EC-1 cell were 24.575μg/ml, 56.991μg/ml, 9.361μg/ml, 13.584μg/ml respectively; for TE-1 cell were 23.534μg/ml, 25.862μg/ml, 6.788μg/ml, 8.911μg/ml respectively; for ECa109 cell were 33.990μg/ml, 33.604μg/ml, 19.873μg/ml, 15.227μg/ml respectively. RHNYP could effectively inhibit the proliferation, migration, invasion, cloning ability, cell cycle and reduce apoptosis of four EC cells. PI3K/AKT/mTOR pathway had closely relationship with the occurrence and development of EC and RHNYP could effectively regulate the proteins expression on the pathway. CDCK3, KIF4A and RAD51AP1 genes were negatively correlated with the overall survival of EC patients and RHNYP could significantly inhibit their expression in four EC cells. Conclusion: RHNYP can effectively inhibit EC cell proliferation, migration, invasion and cell cycle, induce apoptosis, which may be related to the regulation of PI3K/AKT/mTOR pathway. Citation Format: Fuchun Si, Wenyu Zhao, Gao Si, Xuejie Song. Effects of removing heat and nourishing yin prescription on malignant phenotype and PI3K/AKT signaling pathway of human esophageal carcinoma cells [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4720.