Bowen Liu,1 Cong Wang,1 Pengxiang Chen,1 Bo Cheng,2 Yufeng Cheng1 1Department of Radiation Oncology, Qilu Hospital of Shandong University, Jinan, Shandong, People’s Republic of China; 2Department of Radiation Oncology, Shandong Provincial Cancer Hospital, Jinan, Shandong, People’s Republic of China Introduction: Accumulating evidence indicates that RACK1 is involved in the progression of tumors. We aimed to evaluate the function of RACK1 in esophageal squamous cell carcinoma (ESCC) and its role in the mechanism of chemotherapy resistance. Materials and methods: Transfected ESCC cell lines with plasmids expressed shRACK1 or open reading frame (ORF) targeting RACK1 and established stable cell lines. We then examined the effects of RACK1 on cell proliferation and chemotherapy resistance in ESCC cell lines, and the expression of AKT, pAKT, ERK1/2, Bcl-2, and Bim was introduced to further detect the association between RACK1 and chemotherapy resistance. Results: The proliferation ability of ESCC cells was improved in the overexpression RACK1 groups (P<0.001) and decreased in the transfected shRACK1 groups (P<0.001) compared with the control ones. Meanwhile, upregulation of RACK1 significantly suppressed cisplatin-induced apoptosis in Eca109 and EC9706 cells, while downregulation of RACK1 promoted the sensitivity compared to the control group (Eca109: P<0.001 for shRACK1, P<0.01 for shNC, and P<0.001 for overexpression group; EC9706: P<0.001 for shRACK1, P<0.001 for shNC, and P<0.05 for overexpression group). Furthermore, we found that RACK1 could activate the PI3K/AKT pathway and increase the expression level of Bcl-2 in ESCC, which leads to the enhancement of chemoresistance in ESCC. Conclusion: RACK1 promotes proliferation and chemotherapy resistance in ESCC by activating the PI3K/AKT pathway and upregulating the Bcl-2 expression. Keywords: RACK1, ESCC, chemotherapy resistance, PI3K/AKT pathway, Bcl-2