Sproutys (Sprys) are novel negative modulators of mitogen-activated protein kinase (MAPK) involving in growth factor-mediated tyrosine kinase receptor signaling, and have been suggested to be an anti-oncogene as reduced expressions of Sprys have been shown in several cancers. Among four of Spry orthologs the expression of Spry1, 2, and 4 is widespread in embryos and adults, while that of Spry3 is believed to be more restricted, and most of researches are focused on Spry1, 2. Spry4 can inhibit angiogenesis, however, molecular mechanism of the suppression has not yet been clarified completely, and so far no evidences have been reported in vascular smooth muscle cells (SMCs). In the present study, primary cultured aortic SMCs from Spry4 transgenic mice (produced in the Maine Medical Center Research Institute (MMCRI)) or from wild-type mice were plated in 6-well plates at subconfluent density, and transduced with LacZ (Control), Cre or Myc-tagged mouse Spry4 adenoviruses (provided by MMCRI) at a concentration of 400 virus particles per cell. After overnight incubation with virus, medium was replaced with fresh SmGM-2 and cells were incubated for an additional 24 to 48 hr, then the over-expression of Spry4 in mouse SMCs was determined by RT-PCR and immunoblotting. Cultured mouse SMCs were used from passage 4 to 6 for all experiments. Over-expression of Spry4 could obviously inhibit the resting and ET-1 or PDGF-BB activated Raf-ERK ½ and p38 MAPK signal, down-regulate phosphorylated ERK ½ and p38 levels, decrease phosphorylated CyclinD1 expression and MMP13 mRNA level in primary cultured SMCs. Further, we showed that PI3K/Akt signaling and its down-stream target Foxo1/Foxo3 proteins were negatively regulated by Spry4 over-expression. A scratch wound assay to monitor SMC migration showed that the percent closure of wounds in Spry4 conditional over-expression group was obvious lower than control group after wounding 24 (22.25±4.40% vs. 36.18±5.16%, p<0.05) or 48hr (38.44±5.53% vs. 57.83±9.72%, p<0.05). For proliferation studies, SMCs were incubated with 5-bromo-2-deoxyuridine (BrdU) for 24hr, and then were immunostained with a monoclonal anti-BrdU antibody and counterstained with DAPI to label all nucleated cells, and significantly more BrdU positive stained cells were observed in fields of Spry4 conditional over-expression SMCs than control group, and the data clearly showed a lower proliferation rate for Spry4 conditional over-expression SMCs (23.43±3.9%) than for control group (38.03±5.8%) (p<0.05). The above results demonstrate that Spry4 can inhibit MAPK and Akt double signal transduction pathways and suppress proliferation and migration of SMCs, which probably contribute to modulate angiogenesis and vascular remodeling, and are also very important mechanisms in prevention of oncogenesis.