Long-chain n-3 polyunsaturated fatty acids (omega-3) of marine origin exert anti-inflammatory and hypolipidemic effects. We have shown that omega-3 prevent hepatic insulin resistance in obese mice, depending on AMP-activated protein kinase (AMPK) (1). Metformin is the most prescribed antidiabetic drug. However, the precise mechanism of its action remains unknown. Metformin lowers glycaemia presumably via mild suppression of mitochondrial complex I activity, leading to AMPK activation and suppression of hepatic glucose production (2). However, AMPK-independent effects of metformin were observed as well (3). We investigated whether pre-treatment of obese mice with omega-3 could enhance acute metformin action and whether AMPK is involved. Adult male C57Bl/6 mice were fed a high fat diet (HFD; 35 % lipids wt/wt) for 6 weeks, and then either HFD or HFD with 15 % lipids replaced by omega-3 (HFD-F) for 2 weeks. At the end, fasted mice were given a single dose of metformin (400 mg/kg body weight) or saline (placebo) by oral gavage. After 30 min mice underwent oral glucose tolerance test (OGTT; n=6 in each group) or were euthanized by cervical dislocation to collect liver samples for the AMPK activity assay (n=6 in each group) (1). A similar experiment with a lower dose of metformin (60 mg/kg), was performed to study involvement of AMPK in metformin action using mice lacking α2 subunit of AMPK (AMPKα2-KO) (n=6 each group). Data evaluation was performed by ANOVA. During OGTT, glucose levels at 30th and 60th minute, as well as an area under the glycaemic curve (AUC; marker of glucose intolerance), were decreased (1.7-fold, 2.0-fold and 2.9-fold, respectively; p<0.05) after single dose of metformin compared to the saline-treated mice. Two-week consumption of HFD-F diet lowered AUC compared to the HFD diet-fed mice (1.5-fold, p<0.05). Omega-3 treatment tended to augment the effect of metformin, but the effect of the interaction between omega-3 and metformin was not significant (3.2-fold vs. the HFD group; 1.1-fold vs. the HFD + metformin group). No changes in AMPK activity in liver in response to metformin were detected. No difference between control and AMPKα2-KO group during OGTT was observed. We demonstrated acute dose-dependent hypoglycaemic effect of metformin during OGTT in obese mice. Omega-3 lowered the response to glucose challenge, showing a trend for additive improvement of glucose tolerance when combined with a low dose of metformin. No difference in the response to metformin of control and AMPKα2-KO mice suggests that AMPK is not essential for the acute hypoglycaemic effect of metformin. Possible AMPK-independent interaction between omega-3 and metformin in their effects on glucose homeostasis is possible and requires further characterization.