It has previously been shown that hepatic deletion of NADPH oxidase 4 (Nox4) exacerbates the effects of high-fat feeding. In particular, hepatic Nox4 deletion increases adiposity, impairs hepatic insulin signalling ex vivo, and also promotes glucose intolerance (via a glucose tolerance test) and impaired insulin sensitivity (via an insulin tolerance test). Here, we examined in more detail the cause(s) of insulin resistance in vivo, and hypothesised that hepatic Nox4 deletion would result in whole-body insulin resistance specifically due to impairments in hepatic insulin sensitivity. To test this, male mice with hepatic specific deletion of Nox4 (AlbCre-Nox4fl/fl) and wild-type littermates (Nox4fl/fl) were fed a high-fat diet (~23% energy from fat) for 12wk, beginning at 6-7wk of age. At 18-19wk of age, mice had catheters placed into the left carotid artery and right jugular vein, and 5d post-surgery a hyperinsulinaemic-euglycaemic clamp was performed at two insulin doses (2.5 and 4mU/kg/min) in conscious and unrestrained mice (n=6-9 per group). At an insulin infusion rate of 2.5mU/kg/min, glucose infusion rate (GIR), endogenous glucose appearance (endoRa), and insulin-stimulated glucose disposal rate (IS-GDR) were similar between AlbCre-Nox4fl/fl and Nox4fl/fl mice. In Nox4fl/fl mice, increasing the dose of insulin to 4mU/kg/min increased GIR (22±2 vs. 11±1mg/kg/min for 2.5mU/kg/min insulin, p<0.001), enhanced the insulin-induced suppression of endoRa (75±12 vs. 40±7%, p<0.05), and augmented IS-GDR (12±1 vs. 5±1mg/kg/min, p<0.01). In contrast, increasing the dose of insulin from 2.5 to 4mU/kg/min in AlbCre-Nox4fl/fl mice failed to alter GIR (12±1 vs. 11±1mg/kg/min), an effect that was due to (a) an inability to further increase the suppression of endoRa (53±6 vs. 53±3%), and (b) a failure to increase IS-GDR (5±1 vs. 3±1mg/kg/min). Thus, AlbCre-Nox4fl/fl mice are insulin resistant. Furthermore, our findings indicate that the insulin resistance in these mice is due to hepatic and peripheral effects.