Insulin resistance is a worldwide health problem. This study investigated the acute effects of eicosapentanoic acid (EPA) on glucose homeostasis focusing on the role of free fatty acid receptor 1 (FFAR1) and the chronic effects of fish oil omega-3 fatty acids on insulin resistance. Insulin resistance was induced by feeding mice high-fructose, high-fat diet (HFrHFD) for 16 weeks. In the first part, the acute effects of EPA alone and in combination with GW1100 and DC260126 (FFAR1 blockers) on glucose homeostasis and hepatic phosphatidyl-inositol 4,5-bisphosphate (PIP2) and diacylglycerol (DAG) were investigated in standard chow diet (SCD)- and HFrHFD-fed mice. In the second part, mice were treated with fish oil omega-3 fatty acids for 4 weeks starting at the week 13 of feeding HFrHFD. Changes in the blood- and liver tissue-insulin resistance markers and FFAR1 downstream signals were recorded at the end of experiment. Results showed that EPA increased 0 and 30 min blood glucose levels after glucose load in SCD-fed mice but improved glucose tolerance in HFrHFD-fed mice. Moreover, FFAR1 blockers reduced EPA effects on glucose tolerance and hepatic PIP2 and DAG levels. On the other hand, chronic use of fish oil omega-3 fatty acids increased FBG levels and decreased serum insulin and triglycerides levels without improving the index of insulin resistance. Also, they increased hepatic β-arrestin-2, PIP2, and pS473 Akt levels but decreased DAG levels. In conclusion, EPA acutely improved glucose homeostasis in HFrHFD-fed mice by modulating the activity of FFAR1. However, the chronic use of fish oil omega-3 fatty acids did not improve the insulin resistance.