MAN1 (also known as LEMD3) is an integral protein of the inner nuclear membrane. Recently, mutations in MAN1 have been shown to result in osteopoikilosis, Buschke–Ollendorff syndrome and melorheostosis. We show that the nucleoplasmic, C-terminal domain of human MAN1 binds to Smad2 and Smad3 and antagonizes signaling by transforming growth factor-β (TGF-β). In a yeast two-hybrid screen using the C-terminal domain of MAN1 as bait, eight positive clones were obtained that encoded Smad3. In direct two-hybrid assays, this portion of MAN1 bound to Smad2 and Smad3. In glutathione-S-transferase precipitation assays, the C-terminal domain of MAN1 bound to Smad2 and Smad3 under stringent conditions. Antibodies against MAN1 were able to co-immunoprecipiate Smad2 from cells, demonstrating that they reside in the same complex in vivo. TGF-β treatment stimulated transcription from a reporter gene in control cells, but reporter gene stimulation was significantly inhibited in cells overexpressing MAN1 or its C-terminal domain but not its N-terminal domain. TGF-β-induced cell proliferation arrest was also inhibited in stable cell lines overexpressing MAN1. These results show that the nuclear envelope regulates a signal transduction pathway and have implications for how mutations in nuclear envelope proteins cause different human diseases.