The Myc family regulates cell growth and is implicated in the etiology of many cancers, including neuroblastoma. Mxi1, a MAD family member, inhibits N-Myc activity. Mxi0 is an alternatively spliced variant of Mxi1 with a different first exon (Exon 0) whose function has not been determined. These proteins have differential functions in neuroblastoma: Mxi1 inhibits neuroblastoma cell proliferation, while Mxi0 promotes it. While Mxi1 and Mxi0 are mostly homologous, including their repressive domains, they possess distinct N-terminal exons, suggesting a critical role of Exon 0 in the distinct function of Mxi0. To determine the role of subcellular localization as a mechanism for the differential function of Mxi0, we created GFP-tagged constructs of Mxi1, Mxi0, and Exon 0. After expression in 293T cells, localization of Mxi1, Mxi0, and Exon 0 was detected by immunofluorescence. Examination of subcellular location reveals that Mxi1 resides in the nucleus, while Mxi0 is found primarily in the cytoplasm. Exon 0 also displays cytoplasmic localization, indicating that it contributes to differential localization. We then blocked nuclear export with Leptomycin B (LMB) to assess nuclear/cytoplasmic translocation. LMB treatment resulted in nuclear accumulation of Mxi0, suggesting that Mxi0 cycles in and out of the nucleus in response to appropriate signals. This appears to be mediated by Exon 0, as it also accumulates in the nucleus after nuclear export inhibition. In summary, Mxi0 and Mxi1 exhibit distinct subcellular localization patterns, with Mxi1 residing in the nucleus and Mxi0 found in the cytoplasm. Exon 0 directs the cytoplasmic localization of Mxi0. Finally, nuclear export inhibition leads to nuclear accumulation of Mxi0, suggesting that Mxi0 translocates in response to appropriate signals. A better understanding of how Mxi0 impacts neuroblastoma physiology and how Exon 0 imparts the differential function of Mxi0 may aid in developing more effective targeted therapies for children with neuroblastoma.