An extensive accumulation of the matrix component hyaluronan (HA) has been observed in vascular lesions. HA is thought to confer immunomodulatory actions and to regulate cell proliferation and migration in diseased vessels. HA is extruded into the extracellular space by three transmembrane HA-synthase isoenzymes (HAS1, -2, -3). Objective of the current study was to assess specifically the role of HAS3 in vascular pathologies such as restenosis and atherosclerosis. In HAS3 deficient mice (HAS3-/-) and wildtype controls (WT) neointimal hyperplasia was induced using a carotid artery ligation model. Morphometric analysis of carotid arteries harvested 28 days after ligation revealed that lack of HAS3 mitigates neointimal lesion formation as indicated by a decrease in intimal area (AUC: HAS3-/-, 1.53 x107 ± 0.23 x107 μm3 vs. WT, 2.59 x 107 ± 0.34 107 μm3, n = 9, p < 0.05) and reduced intima/media ratio (HAS3-/-, 0.94 ± 0.13 vs. WT, 1.39 ± 0.13, n = 9, p < 0.05). Supportive in vitro studies suggest that blunted neointimal hyperplasia is mainly due to a reduced migration and to a lesser extent reduced proliferation of vascular smooth muscle cell (VSMC). Next atherosclerosis-prone apolipoprotein E/HAS3 double-deficient (apoE-/-/HAS3-/-) mice were generated and fed with a cholesterol-rich Western type diet for 15 weeks. Analysis of aortic en face preparations stained with Oil Red O revealed reduced plaque burden compared to apoE-/- controls (apoE-/-/HAS3-/-, 6.95 ± 2.66, n = 10 vs. apoE-/-, 11.43 ± 4.06, n = 13, p < 0.05). Plasma lipids (total cholesterol, LDL/VLDL, HDL cholesterol) did not differ between the groups. Plaque composition at the aortic root showed decreased collagen content (Picro Sirius Red staining) but no differences in cell density, VSMC content (alpha Actin staining) and macrophage count (Mac2 staining). In contrast, flow cytometry data point to a reduction in circulating T cells (apoE-/-/HAS3-/-, 28.4 ± 1.2 %, n = 5 vs. control, 41.7 ± 5.1 %, n = 6). Collectively, these findings suggest a role for HAS3 in vascular lesion formation. In neointimal hyperplasia HAS3-dependent HA synthesis may support mainly VSMC migration whereas in atherogenesis additional immunomodulatory actions are proposed.