Activation of PPARalpha in the liver is well known to reduce lipid accumulation and improve insulin action in rodents by promoting fatty acid oxidation. Interesting, activated PPARalpha can also promote de novo lipogenesis (DNL) which has recently been shown [1] to be closely associated with endoplasmic reticulum (ER) stress. As ER stress is reported to canuse insulin resistance by disrupting insulin signaling transduction via the unfolded protein response (UPR) pathways, we sought to investigate whether or not PPARalpha activation may exert its beneficial effects via the amelioration of hepatic ER stress. C57BL/6J mice were fed a high fructose diet (HFru) for two weeks to generate ER stress, DNL, steatosis and insulin resistance in the liver and PPARalpha was activated by the administration of fenofibrate (100 mg/kg/day in diet). Compared with untreated mice, HFru-fed mice treated with fenofibrate displayed significant reductions (all p<0.05, n ≥ 8) in visceral adiposity (0.7±0.1 vs 2.0±0.3 Epi/BW), liver triglyceride (13.4±2.0 vs 22.6±1.0 mmol/g) and glucose intolerance (iAUC: 1097±158 vs 1631±106 mM·90min) during GTT (2.5 g/kg, ip) to the similar levels of the chow-fed mice. Fenofibrate treatment increased the activity of beta-hydroxyacyl–CoA dehydrogenase by 2-fold (p=0.01) and restored the impairments of insulin-stimulated phosphorylation of Akt and GSK3beta in the liver. However, the activated IRE1/XBP1 and PERK/eIF2alpha UPR pathways were both exacerbated in fenofibrate treated HFru-fed mice due to elevated expression of lipogenic enzymes mSREBP1c, ACC, FAS and SCD1. Taken together, our findings indicate that the improved glucose tolerance and hepatic insulin signalling produced by PPARalpha activation is due to the elimination of lipid accumulation by an increased fatty acid oxidation. These effects can override the insult of exacerbated ER stress and increased DNL.