Oxidative stress contributes to many cardiovascular pathologies, including hypertension, atherosclerosis and heart failure. The copper-bis(thiosemicarbazone) complex Copper(II)-diacetyl-bis(N4-methylthiosemi-carbazone) [Cu(II)ATSM] was developed as a hypoxia selective positron emission tomography imaging agent, however, recent studies have highlighted the therapeutic properties of Cu(II)ATSM against oxidative stress in the brain. In this study we determine whether Cu(II)ATSM affords protection against the pro-oxidant effects of angiotensin II (Ang II) in human coronary artery smooth muscle cells (HCASMC) via the multifunctional Parkinson’s-associated protein DJ-1 and the redox sensitive transcription factor NF-E2 related factor 2 (Nrf2). Acute treatment with Cu(II)ATSM (1µM, 30 min) attenuated Ang II (200nM, 4h)-induced superoxide generation (Ang II 1.89 x 105 ± 0.13; Ang II + Cu(II)ATSM 0.77 x 105 ± 0.06 mean light units (MLU)/mg protein; p<0.001, n=4-6) as assessed by L-012 chemiluminescence. Cu(II)ATSM treatment alone (0.88 x105 ± 0.13) did not affect basal superoxide levels (1.02×105 ± 0.11). As DJ-1 acts as a copper chaperone protein for cytosolic superoxide dismutase (SOD1), we determined the intracellular levels of Cu and DJ-1/SOD1 protein interaction. Cu levels, determined by inductively-coupled plasma mass spectrometry, were significantly increased following Cu(II)ATSM treatment (1mM, 30 min, 2.395 ± 0.12 µg/L) compared to vehicle (0.89 ± 0.26, P<0.01, n=4), whilst immunopercipitation demonstrated increased protein interaction between DJ-1 and SOD1 following Cu(II)ATSM (1mM, 1h) treatment (n=6). Furthermore, Cu(II)ATSM treatment (1mM, 1h) significantly increased in SOD1 activity (22.94 ± 3.10 U/mg protein) compared to vehicle (11.75 ± 1.38). Pre-treatment of HCASMC with Cu(II)ATSM (1mM, 12h) attenuated Ang II (200nM, 12h)-induced superoxide generation (0.75 x105 ± 0.02 MLU/mg protein) compared to Ang II alone (1.80 ± 0.13, p<0.001, n=4). Cu(II)ATSM treatment (1mM, 1-4h) time dependently increased Nrf2 nuclear localisation determined by immunofluorescence, and induction of the antioxidant protein heme oxygenase-1 (HO-1) after 12h. However, HO-1 protein expression was attenuated in cells deficient for Nrf2 or DJ-1 following siRNA knockdown. Our findings suggest that DJ-1 plays an important role in mediating protection in HCASMC afforded by Cu(II)ATSM, via upregulation of SOD1 activity, and induction of Nrf2- regulated antioxidant proteins. Hence, Cu(II)ATSM may represent a novel therapeutic strategy against oxidative stress in cardiovascular disease.