Smad proteins are critical intracellular mediators of the transforming growth factor-␤, bone morphogenic proteins (BMPs), and activin signaling. Upon ligand binding, the receptor-associated R-Smads are phosphorylated by the active type I receptor serine/threonine kinases. The phosphorylated R-Smads then form heteromeric complexes with Smad4, translocate into the nucleus, and interact with various transcription factors to regulate the expression of downstream genes. Interaction of Smad proteins with cellular partners in the cytoplasm and nucleus is a critical mechanism by which the activities and expression of the Smad proteins are modulated. Here we report a novel step of regulation of the R-Smad function at the inner nuclear membrane through a physical interaction between the integral inner nuclear membrane protein MAN1 and R-Smads. MAN1, through the RNA recognition motif, associates with R-Smads but not Smad4 at the inner nuclear membrane in a ligand-independent manner. Overexpression of MAN1 results in inhibition of R-Smad phosphorylation, heterodimerization with Smad4 and nuclear translocation, and repression of transcriptional activation of the TGF␤, BMP2, and activin-responsive promoters. This repression of TGF␤, BMP2, and activin signaling is dependent on the MAN1-Smad interaction because a point mutation that disrupts this interaction abolishes the transcriptional repression by MAN1. Thus, MAN1 represents a new class of R-Smad regulators and defines a previously unrecognized regulatory step at the nuclear periphery.The transforming growth factor-␤ (TGF␤) 1 superfamily of cytokines, including TGF␤s, BMPs, and activins, plays important roles in the regulation of various aspects of mammalian embryogenesis and carcinogenesis. The signals initiated by these cytokines are transduced by their receptors and the downstream Smad proteins (1-5). Upon ligand binding, activin or TGF␤ receptor serine/threonine kinase phosphorylates and activates the type I receptor kinase, which then phosphorylates the downstream Smad proteins. The Smad proteins are critical mediators of TGF␤ superfamily signaling. Upon phosphorylation by the activated type I receptor kinases, the receptorassociated R-Smads (Smad2 and Smad3 for TGF␤ and activin; Smad1, Smad5, and Smad8 for BMPs) oligomerize with the common mediator Smad4, translocate into the nucleus where they interact with various transcription factors, bind to DNA, and regulate transcription of downstream genes.The expression and activity of the Smad proteins can be modulated by interaction with various cellular proteins at the plasma membrane or in the cytoplasm and nucleus (2, 6, 7). For example, Smad proteins can interact with various transcriptional co-activators on promoter DNAs to regulate the activation of TGF␤, activin, or BMP target genes (2, 6). The activity or intracellular localization of the Smads can be modulated through binding to adaptors molecules such as SARA (8), Hgs (9, 10), chaperones (11), microtubules (12), or various co-repressors such as Ski, SnoN, SNIP,. O...