Various pancreatic β-cell stressors including cytokines and saturated fatty acids are known to induce oxidative stress, which results in metabolic disturbances and a reduction in insulin secretion. However, the key mechanisms underlying dysfunction are unknown. We investigated the effects of prolonged exposure (24 h) to pro-inflammatory cytokines, H 2 O 2 or PA (palmitic acid) on β-cell insulin secretion, ATP, the NADPH oxidase (nicotinamide adenine dinucleotide phosphate oxidase) component p47 phox and iNOS (inducible nitric oxide synthase) levels using primary mouse islets or clonal rat BRIN-BD11 β-cells. Addition of a pro-inflammatory cytokine mixture [IL-1β (interleukin-1β), TNF-α (tumour necrosis factor-α) and IFN-γ (interferon-γ)] or H 2 O 2 (at sub-lethal concentrations) inhibited chronic (24 h) levels of insulin release by at least 50% (from islets and BRIN-BD11 cells), while addition of the saturated fatty acid palmitate inhibited acute (20 min) stimulated levels of insulin release from mouse islets. H 2 O 2 decreased ATP levels in the cell line, but elevated p47 phox and iNOS levels as did cytokine addition. Similar effects were observed in mouse islets with respect to elevation of p47 phox and iNOS levels. Addition of antioxidants SOD (superoxide dismutase), Cat (catalase) and NAC ( N -acetylcysteine) attenuated H 2 O 2 or the saturated fatty acid palmitate-dependent effects, but not cytokine-induced dysfunction. However, specific chemical inhibitors of NADPH oxidase and/or iNOS appear to significantly attenuate the effects of cytokines, H 2 O 2 or fatty acids in islets. While pro-inflammatory cytokines are known to increase p47 phox and iNOS levels in β-cells, we now report that H 2 O 2 can increase levels of the latter two proteins, suggesting a key role for positive-feedback redox sensitive regulation of β-cell dysfunction.