The dynamics of focal adhesions (FAs) are essential physiological processes involved in cell spreading, metastasis, and regulation of the actin cytoskeleton. FAs are complex structures comprising proteins, such as paxillin and zyxin, which interact with extracellular membranes and influence cell motility and morphology. Although related studies have been reported in various cancers, the function and molecular mechanisms of oral squamous cell carcinoma (OSCC) remain unknown. We investigated the coordination between the actin cytoskeleton and FA proteins, specifically introducing pituitary tumor-transforming gene 1 (PTTG1; also known as PTTG1 regulator of sister chromatid separation, securin) into OSCC. Furthermore, we explored the co-localization of several FAs and PTTG1 through small interfering RNA (siRNA) control or siRNA-vasodilator-stimulated phosphoprotein (VASP) and -PTTG1, examining the mechanisms mediated by the induced changes in OSCC both in vitro and in vivo. The knockdown of VASP and PTTG1 regulates the dynamic actin cytoskeleton, restricting cell protrusion and motility from the front to the rear of OSCC cells. Our findings may provide new insights into how cells interact with each other on the surface of FAs in OSCC, influencing metastatic properties.