We identified IFIX as a new member of the hematopoietic interferon (IFN)-inducible nuclear protein with the 200-amino-acid repeat (HIN-200) family. Six different alternatively spliced forms of mRNA are transcribed from the IFIX gene, which are predicted to encode six different isoforms of IFIX proteins (IFIXα1, α2, β1, β2, γ1, and γ2). The IFIX proteins are primarily localized in the nucleus. They share a common N-terminal region that contains a predicted pyrin domain and a putative nuclear localization signal. Unlike IFIXα and IFIXβ, IFIXγ isoforms do not have the 200-amino-acid signature motif. Interestingly, the expression of IFIX was reduced in most human breast tumors and breast cancer cell lines. Expression of IFIXα1, the longest isoform of IFIX, in human breast cancer cell lines reduced their anchorage-dependent and -independent growth in vitro and tumorigenicity in nude mice. Moreover, a liposome-mediated IFIXα1 gene transfer suppressed the growth of already-formed tumors in a breast cancer xenograft model. IFIXα1 appears to suppress the growth of breast cancer cells in a pRB- and p53-independent manner by increasing the expression of the cyclin-dependent kinase inhibitor p21CIP1, which leads to the reduction of the kinase activity of both Cdk2 and p34Cdc2. Together, our results show that IFIXα1 possesses a tumor-suppressor activity and suggest IFIXα1 may be used as a therapeutic agent in cancer treatment.