The association of dysfunctional TGF-β/Smad signaling with aortic diseases is well-established. A variant in the linker domain of SMAD3 (Tyr226Ser) was recently identified in an Icelandic family with high risk of aortic aneurysm. In this study, the molecular and functional effects of the linker domain mutation were investigated in HEK293T cells and endothelial cell lines. Our findings revealed that mutant SMAD3 exhibits a significantly decreased transcriptional activity from the SMAD2/3-specific CAGA promoter upon stimulation with TGF-β. In contrast, a significant increase of transcriptional activity was observed from the SMAD binding element. Furthermore, the SMAD3 mutant was enriched in the nucleus of both HEK293T and endothelial cells when compared to its wild type counterpart. Interestingly, ID-1 mRNA was depleted in the presence of the mutant, whereas an increase of TSP-1, POSTN and LOXL1 expression was observed. Finally, the SMAD3 mutant strongly impaired tube formation of HUVECs. In conclusion, the Tyr226Ser mutation clearly affects the nuclear shuttling of SMAD3 as well as the expression of various extracellular matrix components, which might explain the observed defects in endothelial tube formation.