Epidermal growth factor receptor (EGFR) blockade is a promising therapeutic approach for gastric cancer overexpressing EGFR. EGFR, with a cytoplasmic domain substituted by enhanced green fluorescent protein (DNEGFR-EGFP), can act as a dominant negative mutant receptor to block the EGFR signaling pathway by competing with endogenous EGFR for ligands. The aim of this study was to investigate the effects of DNEGFR-EGFP on the growth, invasion and angiogenesis of human gastric cancer cells, and to elucidate the possible mechanisms behind them. Using multiple cellular and molecular approaches such as gene transfection, MTT, flow cytometry, Western blotting, ELISA, invasion and angiogenesis assays, we found that DNEGFR-EGFP led to G0/G1 arrest by down-regulating cyclin D1 and CDK2 and up-regulating p27, and repressed the invasion and angiogenesis of SGC-7901 cells by inhibiting them from secreting MMP-2, MMP-9 and VEGF. These results indicate that the EGFR blockade strategy (termed dominant negative strategy targeting EGFR) may serve as a promising therapy for the treatment of EGFR-overexpressed gastric cancer.