To investigate the anti-tumor effect and mechanisms of ailanthone (AIL) in tongue squamous cell carcinoma (TSCC). The viability and apoptotic cell number of TCA8113 and Cal-27 cells declined and increased considerably following AIL. Hoechst 33258 staining revealed chromatin aggregation after exposure to AIL. Along with an accumulation of cleaved caspase-9, caspase-3, and PARP1, Bcl-2/Bax ratio and the levels of caspase-3, caspase-9 and PARP1 reduced after exposure to AIL treatment. Subjecting Cal-27 cells to AIL treatment led to the arrestment of the cell cycle at the G2/M phase. Nevertheless, the cell cycle of AIL-treated TCA8113 cells did not change significantly. Following AIL treatment, a decline in the expression of CDK1 and cyclin B1 was manifested by Western Blot. The p-AKT as well as the expression level of p-PI3K underwent a significant downregulation by AIL in both cells. The outcome furnishes a valuable understanding of the potential applications of AIL in treating TSCC.