Transforming growth factor‐β (TGFβ) is a potent regulator of angiogenesis affecting proliferation, differentiation and migration of endothelial cells. The effect of TGFβ on endothelial cells depends on the origin of the cells and on the experimental conditions. Global analysis of TGFβ signalling is expected to unveil mechanisms of this variability and identify novel targets of the growth factor. Here, we report proteome profiling of human microvascular endothelial cells obtained from dermis, which were treated with TGFβ1 and compared to nontreated cells. We identified 54 proteins affected by TGFβ1 using two‐dimensional gel electrophoresis and peptide mass fingerprinting. Thirteen of the identified proteins are involved in various signalling processes. Seven proteins are involved in cytoskeleton rearrangements and six are involved in regulation of metabolism. Ten proteins were identical to predicted hypothetical proteins with no assigned functions. In agreement with the effect of TGFβ1 on components of the cytoskeleton, TGFβ1 induces actin cytoskeleton rearrangements. TGFβ1 also affected expression of E2F6, p57Kip2, G(q)α, hnRNP A1 and myosin light chain proteins as shown by immunoblotting. Down‐regulation of the transcriptional repressor E2F6 by TGFβ1 correlated with a weak growth‐inhibitory activity of TGFβ1 on HMVEC‐d cells. Twenty‐five of the identified proteins have not previously been described as being regulated by TGFβ1, providing new insights into TGFβ1 signalling in endothelial cells.