The complete gI was cloned from the genomic DNA of Pseudorabies virus (PRV) Ea strain and the DNA sequence was determined by Sanger's sequencing technique. The nucleotide and deduced animo acids sequences indicated that the gI gene of PRV Ea strain is composed of 1101 base pairs and could encode 366 animo acids residues. The result of predicted II class structure showed that gI is a typical I class envelope protein. When compared with PRV Rice strain, there were multiple mutations in the gI gene of PRV Ea strain, and the diversity of amino acid residues also existed, especially the deletion of two bases which results in the change of the reading frame and there were additional 16 animo acids residues in the potential cytoplasmic domains of Ea strain. The fragment containing the complete gI gene was further sub-cloned into the downstream of CMV promoter of eukaryotic expression vector pcDNA3.1+, resulting in a recombinant expression plasmid pSB209. pSB209 was transfected into PK-15 cell lines and the cells expressing gI were obtained in the selection of G418. Expressed protein was further detected by Indirect Immunofluorescence Assay(IFA). In order to explore the function of gI in the viral replication, the attenuated live vaccine strain, Bartha in which the gI is deleted, was chosen to determine the plaques forming units (pfu) and the tissue culture infectious dose(TCID50) in PK-15 cell lines expressing the gI(PKgI). The data showed that the pfu and TCID50 in PKgI were 164% and 200% of those in control cell lines respectively. The above results indicated that the gI involve in and accelerate the viral replication.