A hallmark of renal cell carcinoma (RCC) invasion is the degradation of the extracellular matrix (ECM) by the local production of gelatinase enzymes. Matrix metalloproteinase-9 (MMP-9)-induced cancer cell invasion is one of the pivotal steps in cancer metastasis. It has been reported that tumor necrosis factor-α (TNF-α), a regulator of MMP-9, can induce invasion in human renal carcinoma cells. Previous work in our laboratory has shown that rLj-RGD3, a recombinant RGD (Arg-Gly-Asp)-toxin protein from the buccal gland secretion of Lampetra japonica, possesses anti-tumor activity. In this study, we demonstrated that rLj-RGD3 suppressed TNF-α-induced MMP-9 secretion in 786-0 cells (human renal carcinoma cells). To investigate the regulatory effect of rLj-RGD3 on TNF-α-induced MMP-9 secretion, we pre-treated cells with rLj-RGD3. Interestingly, rLj-RGD3 had no significant effect on the constitutive secretion of MMPs. However, low concentrations of rLj-RGD3 decreased TNF-α-induced MMP-9 secretion. Functional studies revealed that rLj-RGD3 induced apoptosis and significantly inhibited the proliferation, migration, and invasion of 786-0 cells. Furthermore, the actin architecture in cells pre-treated with rLj-RGD3 was aggregated and disorganized. Our findings suggest that rLj-RGD3 may be used as a potential drug in renal cancer therapy.