Endoplasmic reticulum stress triggers the activation of unfolded protein response (UPR) pathways is implicated in hepatic de novo lipogenesis (DNL) and insulin resistance. The present study aimed to interrogate the role of each of the three arms of UPR pathways at the onset of hepatic DNL and insulin resistance. To this end, mice were fed one single meal of high fructose (HFru) diet to capture the onset of hepatic DNL. One meal of HFru feeding induced significant increases in the lipogenic enzymes ACC, FAS and SCD1 (by 2-2.5 fold, all p<0.01) compared to chow (CH) feeding. The IRE1/XBP1 arm was significantly activated as indicated by increases in phospho-IRE1 (by 2-fold) and its downstream effector sXBP1 (by 2.5 fold, both p<0.01), while no significant changes were detected for the PERK/eIF2a and the ATF6 arms (p>0.05). Along with these changes, the triglyceride content was significantly elevated (by 2-fold, p<0.05) and the phosphorylation of Akt in response to insulin stimulation (2U/kg, ip) was blunted by ~30% (p<0.05). In contrast, there was no activation of the IRE1/XBP1 arm following one single meal of high fat (HFat) diet or any increase in those lipogenic enzymes (both p>0.05 vs CH), despite similar degrees of hepatic steatosis and reductions in insulin-stimulated Akt phosphorylation (both p<0.05). However, neither of the diets caused a significant impairment in glucose tolerance or visceral adiposity. These data pinpoint the IRE1/XBP1 branch as the key UPR signaling arm involved in the onset of hepatic DNL and this may contribute to the development of hepatic steatosis and insulin resistance in response to HFru diet. Our findings also show that hepatic steatosis and the associated impairment in insulin signaling are early events of the metabolic syndrome induced by HFru or HFat diet prior to the appearance of glucose intolerance and obesity.