Abstract: A recombinant adenovirus expressing hemagglutinin (HA) gene of swine influenza virus (SIV) was constructed by homologous recombination technique. HA gene was subcloned into the vector pAdenoVator-CMV5-IRES-GFP. Then the shuttle plasmid was cotransformed into E. coli BJ5183 with adenovirus backbone plasmid pAdenoVatorΔE1/E3 to generate a recombinant plasmid pAd-HA-GFP through homologous recombination. The plasmid was transferred into 293 cells for packing a recombinant adenovirus. The recombinant adenovirus expressing HA gene, named rAd-HA-GFP, was confirmed by the expression of green fluorescent protein (GFP) and westernblot analysis of HA protein. A single intraperitoneal injection (i.p.) of rAd-HA-GFP was conducted in Kunming mice, a high titer of HI antibody was detected at two weeks after the injection, and the antibody lasted at least for 12 weeks. The results showed that the HA protein expressed by rAdHAGFP possess a good immunogenicity.