Objective: To isolate and clone the interferon regulatory factor（vIRF） encoding K9 gene of human herpesvirus 8 （HHV-8）， and to investigate the expression in the eukaryotic cells. Methods:A pair of PCR primers for K9 gene with HindⅢ and BamHⅠ restriction enzyme cut sites were designed according to the sequence registered in GenBank. Then the genome of primary effusion lymphomas（PEL, also termed body cavity-based lymphomas or BCBL） was taken as template and K9 gene was amplified using PCR. Subsequently, amplified gene fragments were digested with the two enzymes mentioned above and then cloned into pcDNA3.1（+） vector to create recombinant eukaryotic expression plasmid designated as pK9. To facilitate the detection of protein, another pair of PCR primers was designed with the same forward primer and Flag sequence was introduced at the 5′ end of the reverse primer. Then K9-Flag fusion gene was amplified by using PCR, taking plasmid pK9 as template and were further cloned into pcDNA3.1（+） vector to construct recombinant eukaryotic expression plasmid designated as pK9F. After identification with enzyme digestion and nucleotide sequences analysis, recombinant pK9F was transfected into NIH3T3 cells. The expression of pK9F mRNA and protein in NIH3T3 cells was detected by RT-PCR and western blot, respectively. Results: Nucleotide sequences analysis indicated that the isolated and cloned K9-Flag sequence length was 1 380 bp. The isolated K9 sequence was 100% homology with K9 gene previously registered in GenBank. The specific bands were both detected at the expected place by RT-PCR and Western blot. Conclusion: K9 gene could be correctly expressed in NIH3T3 cells.