The development of invasion and metastasis is a major impediment to the successful treatment of cancer. An increase in fibronectin (FN) expression in human tumor has been shown to be correlated with a high potential of tumor cell invasion and elevated matrix metalloproteinases (MMPs) secretion. We have previously shown that human A431 III sub-line isolated from the parental A431 cell (A431P) using Boyden chamber secreted higher levels of MMP-9 than A431P. The A431III cells exhibit elevated migratory, adherent, and spreading characteristics. However, the molecular mechanisms of highly metastatic features in A431III cells have not been fully understood. In this study, we observed that the amounts of endogenous FN in A431III sub-line were significantly higher than those in A431P cells. Moreover, we found that knock-down of endogenous FN by siRNA resulted in totally inhibition of the invasive potential and decreased the ability of wound healing in A431III cells. Since tissue transglutaminase (TG2) has been found to play an important role in tumor progression and could also act as a coreceptor for integrin-mediated binding of cells to FN, we further planed to investigate the roles of TG2 and its relationship with integrins in the potentials of cancer cell invasion and metastasis in highly invasive A431 cancer cell models. On contrary to other previous studies that showed that a decrease of TG2 expression and activity was accompanied by the increasing metastatic potential. Interestingly, our study showed that TG2 levels were significantly increased in A431III cells compared to A431P cells. Furthermore, the interactions of FN, TG2, and integrin β1/β3 were markedly higher in A431III than in A431P cells. After the knockdown of TG2, the invasive ability, MMP-9 secretion, the interaction between FN and integrin β1, FAK phosphorylation, and highly metastasis-related appearances of A431III were decreased to the levels as that of A431P cells. Thus, our results indicate that increased expression of TG2 could enhance association of FN with integrins and trigger integrin-mediated outside-in signaling, at least in part leading to enhancement of the metastatic potentials in A431III cells. Our data suggest that the highly invasive A431III sub-line would be an excellent model for investigating the mechanism of tumor metastasis through FN and TG2-mediated signaling.