Objective To construct an adenovirus vector expressing small interfering RNA (siRNA) targeting to rat Raf-1 gene and identify its function in cardiomyocytes.Methods The siRNA containing DNA sequence targeting to Raf-1 and its negative control sequence were designed, synthesized, annealed and subcloned into adenoviral shuttle vector pAdTrack-CMV. The recombinant adenovirus vector pAd-siRaf-1 was obtained by homologous recombination with pAdTrack-siRaf-1 linearized by PmeI and pAdeasy-1 in bacteria BJ5183, then transfected into HEK293 cells to package the adenovirus. Cardiomyocytes were infected with the adenovirus pAd-siRaf-1, and the expressions of Raf-1 and NF-κB protein were detected by Western blotting.[³H]-leu incorporation was evaluated by scintillation. The surface area of cardiomyocytes was measured using a HJ2000 image analysis system. Results The adenovirus vectors were verified by enzyme digestion and DNA sequencing. Compared with the Ang Ⅱ group, Raf-1 and NF-κB expression, the surface area and [³H]-leu incorporation of cardiomyocytes were significantly decreased in cardiomyocytes infected with the adenovirus PAd-siRaf-1. Conclusions A recombinant adenovirus vector containing rat siRaf-1 gene is successfully constructed. It can effectively reduce Raf-1 and NF-κB expression and cardiomyocyte hypertrophy induced by Ang Ⅱ.