Ginsenoside 20(S)-Rg3 is identified as an active saponin monomer which derived from red ginseng and is demonstrated to play an anti-tumor role in diverse cancers. MicroRNAs (miRNAs) are important regulators in the progression of cancers, containing esophageal squamous cell carcinoma (ESCC). It was reported that microRNA 324-5p (miR-324-5p) exerted critical functions in some cancers; however, the detailed molecular mechanism of miR-324-5p mediated by 20(S)-Rg3 to suppress cell viability in ESCC has not been explored. Herein, we explored the function of 20(S)-Rg3 or miR-324-5p on ESCC cell viability by MTT assay, colony formation assay, flow cytometry analysis and western blot analysis. The binding of miR-324-5p to its target gene, proteasome activator subunit 3 (PSME3), was confirmed through RNA pull down and luciferase reporter assays. The results indicated that 20(S)-Rg3 significantly inhibited cell viability and the cell cycle and facilitated cell apoptosis. Furthermore, this effect was strengthened with the increased concentration of 20(S)-Rg3. Moreover, we found that miR-324-5p level was increased under 20(S)-Rg3 treatment. Additionally, overexpressed miR-324-5p inhibited ESCC cell viability, and downregulated miR-324-5p recovered inhibited cell viability caused by 20(S)-Rg3. Further exploration verified that miR-324-5p targeted PSME3, and PSME3 deficiency countervailed the effect of miR-324-5p inhibition on ESCC cell viability under 20(S)-Rg3 treatment. Conclusively, 20(S)-Rg3 suppresses cell viability in ESCC via mediating miR-324-5p-targeted PSME3.