Objective: To investigate the effect of MMP-9 inhibitor (Mki67) on the biology of human oral squamous cell carcinoma SCC15 cell line and to explore its mechanism of action through PI3K/Akt signaling pathway. Methods: SCC15 cells were extracted, and the supernatant was discarded. The cells were then rinsed twice with PBS, and 0, 2.5, 5, and 10 μL of Mki67 (50 mg/mL) were added to the culture respectively. The inhibition rate of cell proliferation was detected by 3-(4, 5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) method, and the cell migration was measured by Transwell chamber test. The cell apoptosis rate was detected by cytometry, and the p-Akt protein content in the cells of each group was determined by a double-antibody sandwich enzyme-linked immunosorbent assay (ELISA) kit. Results: The cell proliferation rates of the 2.5 μL, 5 μL, and 10 μL dose groups were all lower than the 0 μL group (P < 0.05) before treatment, and the cell proliferation rates in the 2.5 μL, 5μL, and 10μL dose groups decreased overtime (P < 0.05). After 24 h, with the increase of Mki67 concentration, the number of migration and invasion gradually decreased (P < 0.05), and the number of apoptosis gradually increased (P < 0.05); besides, the relative expression of MMP-9, PI3K, and Akt mRNA decreased gradually (P < 0.05), and the expression level of Akt mRNA was not statistically significant (P > 0.05). Conclusion: MMP-9 inhibitor (Mki67) can inhibit the proliferation and migration of SCC15 cell line and induce apoptosis, and its mechanism of action may be related to the inhibition of PI3K/Akt signaling pathway.