P21 activated kinase 4 (PAK4), as an effector of Cdc42, playing important roles in regulating the processes of cytoskeleton organization. PAK4 has been considered to be an oncogenic protein, which has strong relationship with gastric cancer metastasis. However, the mechanism of PAK4 in regulating gastric cancer metastasis is still not fully understood. In this study, using yeast two-hybrid system, we identified that the eukaryotic elongation factor 1 α1 (eEF1A1) is a new binding partner of PAK4. The interaction between PAK4 and eEF1A1 was confirmed by GST pull-down and co-immunoprecipitation. PAK4 co-localized with eEF1A1 in the cytoplasm of gastric cancer cells. Overexpression of PAK4 enhanced the expression level of eEF1A1 and vice versa. PAK4 and eEF1A1 could cooperate to promote gastric cancer cell migration and invasion. Furthermore, the expression of PAK4 and eEF1A1 in clinical gastric cancer samples were examined by western blotting and immunohistochemistry. Statistical analysis indicated that there was positive correlation between the expression of PAK4 and eEF1A1. This study demonstrated for the first time that PAK4 interacted with eEF1A1 to promote migration and invasion of gastric cancer cells, thereby providing new insights into the function of PAK4 and eEF1A1 in the progression of gastric cancer.