Enhanced oxidative stress appears as a common pathogenic mechanism of vascular diseases, like coronary artery disease or diabetic vasculopathy. Increased vascular activity of the superoxide anions–releasing enzyme xanthine oxidase (XO) has been reported in such diseases. Mitogen-activated protein kinases (MAPK) are key signalling enzymes that regulate growth, inflammation and cell death in eukaryotic cells. We analysed the role of p38 MAPK and JNK, which can be activated by cellular stress, inflammatory cytokines and some mitogenic molecules as mediators of XO signalling in cultured human aortic smooth muscle cells (HASMC), obtained by enzymatic dissociation from five organ donors. Treatment of HASMC with 100 μM xanthine together with XO (50 μU/ml) resulted in increased intracellular content of superoxide anions, as shown with the fluorescent probe dihydroethidine, which could be prevented by both superoxide dismutase (SOD; 100 U/ml) and the specific XO inihibitor allopurinol (100 μM). In addition, XO activated both p38 and JNK MAPK, with maximal effects at 10 (1.50±0.48 times vs time 0) and 30 min (1.77±0.21 times vs time 0), respectively. XO-induced MAPK activation was not observed upon co-incubation with SOD or allopurinol. XO also stimulated both de novo synthesis (151.47±35.43% vs time 0) and activity of the transcription factor AP-1 (2.24±0.27 times vs control). The p38 inhibitor SB203580 (5 μM), but not the JNK inhibitor SP600125 (10 μM), significantly reduced increased AP-1 levels. Finally, XO increased HASMC size (117.52±3.01% vs control) without affecting proliferation through a SOD-dependent mechanism. This hypertrophic effect of XO was dependent on JNK and p38 activation, as it was prevented by both SB203580 and SP600125. In summary, XO can favour, through MAPK signalling, an activated pro-inflammatory and growth-promoting state of human VSCMC, which may in turn contribute to the development and maintanance of vascular diseases.