Our previous study suggested that receptor for activated C kinase 1 (RACK1) contribute to the progression of oral squamous cell carcinoma (OSCC). The aim of this study is to elucidate the mechanism by which RACK1 regulates cell growth in OSCC using in vitro and in vivo models. The effects of RACK1 knockdown with lentivirus based shRNA in stable cell lines were evaluated by Q-PCR and western blot analysis. RACK1 silencing effects on the cell cycle in OSCC cells were detected by flow cytometry and western blot analysis. The effect of RACK1 silencing on inhibiting the progression of OSCC was illustrated using a xenografted mouse model. RACK1 and relevant signaling pathways were investigated in tissues and cells using immunohistochemistry and/or western blot analysis. Stable silencing of the RACK1 gene resulted in a distinct G1 and G2 phase arrest by downregulating Cyclin B1 and Cyclin D1. Depleted RACK1 led to markedly decreased tumor volume and the expression of Ki67, CD34, and VEGF in vivo. The expression of RACK1 and p-AKT has a parallel pattern in different stages of oral carcinogenesis tissues. In addition, the protein level of RACK1 was positively correlated with p-AKT in OSCC tissue samples and cell lines. We found specific transient knockdown of RACK1 could downregulate the protein levels of p-AKT, p-mTOR, and p-S6 in a dose-dependent manner. This study demonstrates that RACK1-dependent OSCC growth and survival may be related to the increased activation of the AKT/mTOR/S6 pathway.