Vav3, a Rho GTPase guanine nucleotide exchange factor, is associated with tumor growth, apoptosis, invasion and metastasis, and angiogenesis. However, the role of Vav3 in gastric cancer remains unclear. In this study, Vav3 expression was blocked by specific siRNA in gastric cancer cell line MGC803. MTT was used to assay cell proliferation activity; wound healing assay and transwell assay were applied to detect cell migration and invasion ability; and qRT-PCR and Western blot were employed to detect expression levels of Vav3 as well as proliferation, migration, and invasion-related genes. The results showed that Vav3 expression in gastric cancer tissues and cell lines was significantly upregulated and was higher than that in adjacent tissues of cancer and normal gastric mucosal cell lines. Vav3 knockdown inhibited proliferation, migration, and invasion of MGC803 gastric cancer cells. The expression of P21, P27, TIMP-1, and TIMP-2 was upregulated, while proliferating cell nuclear antigen, cyclin E1, matrix metalloproteinase (MMP)-2, and MMP-7 were downregulated by Vav3 knockdown in MGC803 gastric cells. In conclusion, Vav3 is involved in the proliferation, migration, and invasion of gastric cancer cell as a tumor oncogene.