The invasion of Staphylococcus aureus into alveolar epithelial cells is regarded as the key step for S. aureus lung infection. However, the mechanism of internalization of S. aureus by alveolar epithelial cells is not clear, and was the aim of this investigation Human lung adenocarcinomic epithelial cells and A549 cells were used. Human β1 integrin and rat β1 integrin were detected by real-time reverse transcription (RT)-PCR. The expressions of β1 integrin, Akt and p-Akt were detected by Western blot analysis. To further investigate the role of β1 integrin in S. aureus internalization by alveolar epithelial cells, we next performed siRNA-mediated knockdown of β1 integrin expression. In this study, we found that S. aureus invades human alveolar epithelial cells and rat primary alveolar epithelial cells. The β1 integrin ligand competitive inhibitor, GRGDS-peptide, blocked the internalization of S. aureus by A549 cells. Knockdown of β1 integrin also inhibited the internalization of S. aureus. In addition, the PI3K/Akt signaling pathway in alveolar epithelial cells was activated by the infection with S. aureus. Furthermore, Akt phosphorylation was abolished by transient transfection with β1 integrin siRNA in A549 cells challenged with S. aureus. Our results suggest that the phosphatidylinositol 3-kinase/Akt signaling pathway plays an important role in β1 integrin-mediated internalization of S. aureus by alveolar epithelial cells.