Interleukin-6 (IL-6) is a multifunctional cytokine that is associated with the disease status and outcomes of gastric cancer. Nonetheless, the underlying mechanism of how IL-6 promotes the spread of gastric cancer is still unclear. In this study, we used a modified Boyden chamber assay to test the invasion ability of different gastric cancer cell lines. Liposome-mediated transfection was used to introduce an IL-6 expression vector into AGS cells, and the transfectants were further examined for the expression of active RhoA and phosphorylated Src using a pull-down assay and coimmunoprecipitation/Western blot analysis. Furthermore, RhoA expression in gastric adenocarcinoma specimens was investigated immunohistochemically. We documented that IL-6 could promote AGS cell motility and invasiveness, and inhibition of RhoA expression by dominant negative RhoA, C3 transferase, or dominant negative Src expressing plasmids could effectively decrease the invasiveness of IL-6 transfectants. We also documented an interaction between active RhoA and phosphorylated-Src following IL-6 treatment. Gastric cancers displaying high expression of RhoA are highly correlated with aggressive lymph node metastasis, more advanced tumor stage, histologically diffuse type and poorer survival. In conclusion, IL-6 induces AGS gastric cancer cell invasion via activation of the c-Src/RhoA/ROCK signaling pathway and RhoA expression could be used as a prognostic factor in patients with gastric adenocarcinoma.