Abstract: Background and Objective: Squamous cell carcinoma is a malignant tumor derived from epithelial tissue with cell structure group, capable to infiltrate through the bloodstream and lymphatic tissue, spreading throughout the body. This study aim to complete theoretical foundation of flavonoid compound from anthill plant (Myrmecodia pendans) which contribute in growing cell line oral tongue squamous cell carcinoma through proliferation inhibition, inhibition mechanism transduction Akt signal and NF-κB in tongue cancer cell Supri’s-clone (SP-C1). Application benefit to explore potential fractionation anthill plant use herbal ingredients for chemo protective therapy. Materials and Methods: This whole study conducted with experiment laboratorium method utilized tongue cancer human cell SP-C1. This study consist 2 steps, first to determinate, extraction and fractionation anthill plant and carry out tonicity test to get flavonoid fraction from anthill plant which has anticancer potential against tongue cancer cell SP-C1. The second stage held with invasion inhibition test, proliferation and inhibition test against protein Akt expression and NF-κB in tongue cancer cell SP-C1. The barriers to proliferation through the test of MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay, barriers to invasion through Boyden chamber assay and the membrane polycarbonate, ELISA analysis and Western blotting analysis on the obstacle Akt signal transduction pathways and transcription factor of nuclear factor-kappa B (NF-κB). Data analysis conducted with 2 way ANOVA followed with LSD post hoc test with significance is set on 95%. Pearson correlation conduct to find strong relationship intervariable. Results: This study showed that the average cell growth inhibition SP-C1 based on the time and concentration using the MTT [3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assay. The resistance of cancer cell in vitro SP-C1. On ELISA testing and Western blotting analysis, inhibiton of protein expression of Akt signal transduction and transcription factor of nuclear factor-kappa B (NF-κB) showed increased protein expression was significantly obstacles and prove that the ethyl acetate fraction flavonoid inhibits translocation and activation of transcription pathway NF-κB and growth factors that induces the phosphorylation of Akt signal transduction pathway. Conclusion: Ethyl acetate fraction flavonoid anthill has antitumor activity in multiple molecular targets transduction pathway including Akt and nuclear factor-kappa B (NF-κB) squamous cell carcinoma of the tongue.