Background Recent clinical trials have demonstrated a role for sodium glucose cotransporter 2 (SGLT2) inhibitors in improving cardiovascular outcomes in heart failure patients, but the underlying mechanisms remain unknown. We investigated the direct effects of canagliflozin, a non-selective SGLT1/SGLT2 inhibitor on myocardial redox signalling in humans. Methods Study 1 included 364 patients undergoing cardiac surgery. Human right atrial appendage biopsies, obtained during surgery, were used to quantify the sources of superoxide (O2.-) and the gene expression of inflammation, fibrosis and myocardial stretch markers. In Study 2, myocardial biopsies from 51 patients were used ex vivo to study the direct effects of canagliflozin on O2.- generation and understand its role in controlling the activity of NADPH-oxidases and uncoupled nitric oxide synthase (NOS). Finally, we used differentiated H9C2 and human primary cardiomyocytes (hCM) to further characterise the key regulatory mechanisms (Study 3). Results SGLT1 was abundantly expressed in the human myocardial biopsies and hCM whilst SGLT2 was barely detectable. SGLT1 expression levels were positively correlated with basal O2.- production and the expression of natriuretic peptides, proinflammatory cytokines and pro-fibrotic markers in human myocardial biopsies from study 1. Incubation of human myocardium with canagliflozin significantly reduced basal and NADPH-oxidase-derived O2.- via AMP kinase (AMPK)-mediated suppression of GTP-activation and consequent reduction of membrane translocation of Rac1, an NADPH-oxidase subunit. This resulted in reduced oxidation and increased bioavailability of tetrahydrobiopterin, the nitric oxide synthase (NOS) co-factor essential for enzymatic coupling, leading to improved NOS coupling. These findings were replicated in hCM, where canagliflozin was shown to regulate AMP/ATP ratio, which could be upstream of AMPK activation. The effects of canagliflozin were significantly attenuated by knocking-down SGLT1 in hCM. Transcriptional profiling of hCM treated with canagliflozin revealed that canagliflozin had striking effects on myocardial redox signalling, causing suppression of apoptotic and inflammatory pathways in the human heart. Conclusions We demonstrate for the first time in humans that canagliflozin suppresses myocardial NADPH-oxidase activity and improves NOS coupling through an SGLT1/AMPK/Rac1-mediated pathway, leading to global anti-inflammatory and anti-apoptotic effects in the human myocardium. These findings provide a mechanistic basis for the beneficial effects of SGLT1/2 inhibitors in patients with heart failure. Funding Acknowledgement Type of funding source: Foundation. Main funding source(s): 1. British Heart Foundation (FS/16/15/32047 and PG/13/56/30383 to CA, CH/16/1/32013 to KC, and Centre of Research Excellence award RG/13/1/30181), 2. The Japanese Heart Rhythm Society-European Heart Rhythm Association fellowship grant sponsored by Biotronik.