Studies have shown that stress such as hypoxia, chemotherapy, radiotherapy can lead to polyploidization of tumor cells, which play an important role in tumor heterogeneity and malignant phenotype. Paclitaxel (PTX) treatment promoted polyploid cancer cells (PCCs) formation, and miR-378d is sharply reduced in PCCs of esophageal squamous cell carcinomas (ESCC) cells, but miR-378d participation PCCs formation and the impact on the biological behavior of ESCC remains unclear. We analyzed the PCCs formation and biological behavior of ESCC cells in vivo and in vitro, and the related proteins regulated by miR-378d. Results showed that miR-378d expression was associated with good prognosis in ESCC patients. miR-378d inhibition promoted PCCs formation, heterogenicity, chemo-resistance, monoclonal formation, EMT, migration, invasion, stemness and metastasis of ESCC cells. miR-378d can target downregulated AKT1, and inactivating the AKT-β-catenin signaling pathway, miR-378d and AKT can also regulated RhoA expression. AKT and RhoA regulated polyploidization and depolyploidization. Therefore, miR-378d expression is a good prognostic factor of ESCC patients and regulates polyploidization and malignant phenotype of tumor cells through AKT and RhoA.