Phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) signaling pathway plays a crucial role in the formation and progression of many malignancies, and has been shown to be an important therapeutic target for cancer. In the present study, human gastric adenocarcinoma tissues of different grades (N=45) were collected. The protein expression of PI3Kp85α and phosphorylated AKT (p-AKT) was evaluated immunohistochemically in the biopsy samples. PI3K/AKT pathway was blocked by constructed recombinant small hairpin RNA adenovirus vector rAd5-PI3Kp85α (rAd5-P) used to transfect into human gastric cancer SGC-7901cell line. The transfection efficiency of rAd5-P in SGC-7901 cells was observed under fluorescent microscope. The expression of PI3Kp85α, p-AKT, Ki-67 and matrix metallopeptidase-2 (MMP-2) was detected by real-time PCR and Western blot assays. Cell proliferative activities and metastatic capabilities were determined by MTT and Transwell assays. As a consequence, the protein expression of PI3Kp85α and p-AKT was respectively observed in 80.0% and 82.2% gastric adenocarcinoma tissues, elevating with the ascending order of tumor malignancy. Targeted blockade of PI3K pathway decreased the expression of PI3Kp85α, p-AKT, Ki-67 and MMP-2, and inhibited the proliferative activities and metastatic capabilities of gastric cancer cells. In conclusion, PI3Kp85α and p-AKT were strongly expressed in gastric adenocarcinoma tissues, and targeted blockade of PI3K pathway may inhibit gastric cancer growth and metastasis through down-regulation of Ki-67 and MMP-2 expression. PI3K/AKT pathway may represent an important therapeutic target for gastric cancer.