Vascular endothelial growth factor 165 (VEGF(165))-mediated autocrine stimulation of tumor cells enhances the progression to a malignant phenotype. VEGF(165)b competes with VEGF(165) and binds to vascular endothelial growth factor receptor (VEGFR), resulting in inhibition of downstream signal transduction pathways. This study was designed to investigate the role of VEGF(165)b in the migration and invasion of human lung adenocarcinoma A549 cells. The full-length of VEGF(165)b was constructed and cloned into an expression plasmid (pVEGF(165)b), and then transfected into A549 cells. Dimethylthiazolyl- 1 -2, 5-diphenyltetrazolium bromide (MTT) assay was used to detect the effect of VEGF(165)b on proliferation of transfected cells. Reverse transcription polymerase chain reaction (RT-PCR) was employed to examine the effect of VEGF(165)b on the expression of VEGF(165) in transfected cells. Wound-healing assays were used to investigate the effect of VEGF(165)b on migration of transfected cells. Matrix metalloproteinase (MMPs) activity assay and in vitro invasion assay were used to determine the role of VEGF(165)b in invasion of transfected cells. There was no significant change in proliferation of A549 cells after transfection of pVEGF(165)b, but the expression of VEGF(165), migration and invasion in A549 cells were inhibited. Furthermore, exogenous VEGF(165)b inhibited the activity of MMP9 in the supernatant of A549 cells and the subsequent invasion capacity of those cells. We therefore conclude that exogenous VEGF(165)b can inhibit the expression of VEGF(165), as well as the migration and invasion of A549 cells, but has no effect on the proliferation of A549 cells.