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Aim: Cytokine receptor-like factor 3 (CRLF3) is a novel gene cloned from K562 cell line. The aim of the current study is to observe CRLF3 expression and subcellular localization in 293T cells and to express and purify the CRLF3 fusion protein in E.coli., Methods: The plasmid pCMV-myc-CRLF3 was transiently transfected into 293T cell. The expression and localization of CRLF3 protein was observed by immunostaining approach. CRLF3 was also cloned into pGEX-4T-1 vector. The GST-CRLF3 fusion protein was expressed in E.coli and purified by GST affinity chromatography., Results: CRLF3 protein was highly expressed in transfected 293T cells. CRLF3 protein was distributed in both cytoplasm and cell membrane. The GST-CRLF3 fusion protein was expressed as a M(r) 74 000 protein and then successfully purified from E.coli cells., Conclusion: Overexpressed CRLF3 is a cytoplasmic protein that distributes in both cytoplasm and cell membrane in mammalian cells. The recombinant GST-CRLF3 fusion protein had been isolated and could be used for further antibody production and functional characterization.