This study was purposed to investigate the expression of ifi56 gene in the ATRA-induced acute promyelocytic leukemia (APL) NB4 cell differentiation and to construct the eukaryotic expression plasmid of ifi56 gene. RT-PCR was used to detect the expression of ifi56 in NB4 cells treated with ATRA for different time. Human ifi56 cDNA was amplified by RT-PCR and cloned into pEGFP-C1 vector, then was transfected into 293T cells. The expression of the recombinant protein in 293T cells was detected by Western blot. The localization of IFI56 protein was observed by fluorescence microscopy. The results showed that the ifi56 mRNA was almost undetectable in untreated NB4 cells, but it significantly increased after ATRA treatment for 72 hours. The cDNA fragment of ifi56 was inserted into the expressing plasmid pEGFP-C1 successfully. The expression of EGFP-IFI56 fusion protein with a molecular weight about 83 kD was detected by Western blot. The EGFP-IFI56 protein was localized in cytoplasm mainly. It is concluded that the expression of ifi56 is enhanced significantly when the differentiation of APL cells was induced by ATRA. Gene ifi56 is successfully cloned into eukaryotic expression vector and the fusion protein is expressed in the cytoplasm mainly.