Secretion of the adipocytokine adiponectin inversely correlates with adiposity. Adiponectin has been proposed to have insulin-sensitising and anti-inflammatory actions in metabolic tissues and anti-atherogenic actions in blood vessels. We have recently demonstrated that adiponectin secretion is reduced in perivascular adipose tissue (PVAT) from mice lacking the α1 catalytic subunit isoform of AMP-activated protein kinase (AMPK), a key regulator of cellular and whole body metabolism. Furthermore, adiponectin attenuated contraction of endothelium-denuded aortic rings in response to the thromboxane A2 receptor agonist U46619, suggesting adiponectin has direct anti-contractile effects on vascular smooth muscle. We therefore investigated whether adiponectin influenced contractile signalling pathways in cultured human aortic vascular smooth muscle cells (HAVSMCs). The effect of globular adiponectin (gAd) on contractile signalling in HAVSMCs stimulated with U46619 was assessed by immunoblotting with specific antibodies to contractile signalling proteins. Intracellular HAVSMC Ca2+ concentrations were assessed using the ratiometric fluorescent dye Fura2. Rho-associated protein kinase (ROCK) activity and actin filament formation were assessed by ELISA and immunofluorescence microscopy of HAVSMCs stained with phalloidin respectively. Data are reported as mean±SEM. Statistical significance was assessed by 2-way ANOVA. U46619 stimulated phosphorylation of myosin light chain (MLC) T18/S19 and the MLC phosphatase subunit MYPT1 at T696 2.8±0.3-fold (n=4, p<0.0001) and 1.25±0.02-fold (n=3, p<0.001) respectively. Preincubation of HAVSMCs with gAd completely abrogated U46619-stimulated phosphorylation of either protein (p<0.001). MYPT1 has been previously demonstrated to be inhibited by ROCK-mediated phosphorylation at T696, thereby increasing MLC phosphorylation and subsequent contraction. U46619 stimulated ROCK activity 2.3±0.3-fold (n=3, p<0.05), which was inhibited by preincubation with gAd (p<0.05). In contrast, preincubation with gAd had no effect on U46619-stimulated increases in intracellular Ca2+ or protein kinase C (PKC) activation. Furthermore U46619-stimulated actin filament formation 1.9±0.3-fold (n=3, p<0.01), an effect that was inhibited by preincubation with gAd or the ROCK inhibitor Y27632 (p<0.05 in each case). Intriguingly, although gAd has been previously reported to activate AMPK in many cell types, it had no effect on AMPK activity in HAVSMCs. These data indicate that gAd inhibits U46619-stimulated contractile signalling in HAVSMCs by reducing ROCK activity, leading to decreased MYPT1 phosphorylation, increased dephosphorylation of MLC and reduced actin filament formation. The effects of gAd were independent of Ca2+, PKC and AMPK. These data identify a hitherto unappreciated mechanism by which adipose tissue may regulate vascular cell function.