Background: Palmitoleic acid, since described as lipokine, increases glucose uptake by modulation of 5′AMP‐activated protein kinase (AMPK), as well as increasing lipolysis by activation of peroxisome proliferator‐activated receptor‐α (PPARα), in adipose tissue. However, in liver, the effects of palmitoleic acid on glucose metabolism and the role of PPARα remain unknown. Objective: To investigate whether palmitoleic acid improved the hepatic insulin sensitivity of obese mice. Methods: C57BL6 and PPARα knockout (KO) mice were fed for 12 weeks with a standard diet (SD) or high‐fat diet (HF), and in the last 2 weeks were treated with oleic or palmitoleic acid. Results: Palmitoleic acid promoted a faster uptake of glucose in the body, associated with higher insulin concentration; however, even when stimulated with insulin, palmitoleic acid did not modulate the insulin pathway (AKT, IRS). Palmitoleic acid increased the phosphorylation of AMPK, upregulated glucokinase and downregulated SREBP‐1. Regarding AMPK downstream, palmitoleic acid increased the production of FGF‐21 and stimulated the expression of PPARα. Palmitoleic acid treatment did not increase AMPK phosphorylation, modulate glucokinase or increase FGF‐21 in liver of PPARα KO mice. Conclusions: In mice fed with a high‐fat diet, palmitoleic acid supplementation stimulated the uptake of glucose in liver through activation of AMPK and FGF‐21, dependent on PPARα. J. Cell. Physiol. 232: 2168–2177, 2017. © 2016 Wiley Periodicals, Inc. Palmitoleic acid (PM) increased glucose uptake and modulated glucokinase in liver; Those effects are linked to higher PPARα expression, activation of AMPK and FGF‐21. Similar beneficial effects were not observed in liver of PPARα KO mice. Thus, activation of AMPK and FGF‐21 by PM in liver, are dependent of PPARα.