Objective：To clone mouse Marf1 gene and let it ectopically express in HEK293T cells，for studying its expression and mechanism. Methods：Marf1 coding sequence was amplified by reverse transcription-polymerase chain reaction(RT-PCR)and was cloned into the eukaryotic cell-expression vector pCMV6-AC-3DDK and pCMV6-AN-mKate using the advanced In-Fusion cloning system. After verification by restriction digestion followed by electrophoresis and sequencing，the correctly cloned plasmid DNA was transfected into HEK293T cells. The expression of MARF1-3DDK and MARF1-mKate fusion protein in the transfected HEK293T cells was assessed by both Western blot and immunofluorescence using the antibodies against DDK and mKate tag proteins. Results：The correct cloning of Marf1 was confirmed by restriction digestion and sequencing，and the expression of MARF1-3DDK and MARF1-mKate fusion protein was detected by Western blot. MARF1 fusion protein was detected to be localized either in the cytoplasm in a punctate manner or on the mitotic spindles. Conclusion：Mouse Marf1 gene was successfully cloned and expressed in HEK293T cells，which lays solid foundation for further studies on its functions and the related mechanisms.