TGF-beta is a ubiquitously expressed cytokine that signals through the Smad proteins to regulate many diverse cellular processes. SnoN is an important negative regulator of Smad signaling. It has been described as a nuclear protein, based on studies of ectopically expressed SnoN and endogenous SnoN in cancer cell lines. In the nucleus, SnoN binds to Smad2, Smad3, and Smad4 and represses their ability to activate transcription of TGF-beta target genes through multiple mechanisms. Here, we show that, whereas SnoN is localized exclusively in the nucleus in cancer tissues or cells, in normal tissues and nontumorigenic or primary epithelial cells, SnoN is predominantly cytoplasmic. Upon morphological differentiation or cell-cycle arrest, SnoN translocates into the nucleus. In contrast to nuclear SnoN that represses the transcriptional activity of the Smad complexes, cytoplasmic SnoN antagonizes TGF-beta signaling by sequestering the Smad proteins in the cytoplasm. Interestingly, cytoplasmic SnoN is resistant to TGF-beta-induced degradation and therefore is more potent than nuclear SnoN in repressing TGF-beta signaling. Thus, we have identified a mechanism of regulation of TGF-beta signaling via differential subcellular localization of SnoN that is likely to produce different patterns of downstream TGF-beta responses and may influence the proliferation or differentiation states of epithelial cells.