Phosphatidylinositol 3-kinase (PI3K)/Akt plays a critical role in the formation of many malignant tumors, and has been shown to be an important therapeutic target. In the present study, small hairpin RNA (shRNA) expression constructs that target sequences of human Akt1 and PIK3R1 were used to examine the proliferation and invasion inhibition effects on SGC7901 gastric adenocarcinoma cells and U251 glioma cells. Cell growth was inhibited by over 60%, as indicated by a MTT assay, and was accompanied by G(1)/G(0) phase arrest in the shRNA treated group, indicating poor cell growth activities. The number of cells invading through the matrigel in the shRNA treated group were significantly decreased (51.6 +/- 3.9) compared with that of the control group (105 +/- 4.0) and the nonsense sequence group (102.5 +/- 6.4). In addition, the tumor volumes in the SGC7901 subcutaneous nude mouse model treated with shRNA were significantly smaller than those of the control group and nonsense sequence group. When Akt1 and PIK3R1 were dramatically downregulated, proliferating cell nuclear antigen (PCNA), CyclinD1 and matrix metalloproteinases (MMP-2, MMP-9) were downregulated, while tissue-Inhibitor of Metalloproteinase-2 (TIMP-2) and p53 were upregulated. Our results demonstrated that shRNA targeting Akt1 and PIK3R1 downregulates their expression significantly in a sequence-specific manner, exerting proliferation and invasion inhibition effects on SGC7901 and U251 cells. In conclusion, our data suggests a novel mechanism for the regulation of malignant tumor cell growth and provides evidence for new combinatory gene therapy for malignant tumors.