We aimed to evaluate activation of macrophages in insulin‐sensitive tissues (liver, adipose tissue, and muscles) under high‐fat diet (HFD) and elucidate the role of Kupffer cells (KC) in HFD‐induced insulin resistance. Tissue macrophage populations, insulin signaling, and sensitivity were evaluated in mice fed a HFD for 4 or 16 wk. Selective KC depletion was obtained by intravenous injections of liposome‐encapsulated clodronate. Mice fed a HFD for 4 to 16 wk have hepatic and peripheral insulin resistance together with macrophage recruitment in the adipose tissue but not in the liver. Depletion of KC for the last 10 d of the 16 wk experiment fails to improve insulin sensitivity compared to PBS‐treated animals. In contrast, preventive KC depletion prior to and during the 4 wk HFD attenuates the development of obesity, adiposity, adipose tissue inflammation (P<0.01 vs. PBS group), and insulin resistance (P<0.01). Interestingly, in mice fed a normal diet, prolonged KC depletion ameliorates insulin sensitivity and decreases adiposity without altering physiological body weight gain or food intake. Preventive and prolonged KC depletion ameliorates insulin sensitivity and prevents adipose tissue inflammation, suggesting a communication between the liver and the adipose tissue in the development of HFD‐induced metabolic alterations.—Lanthier, N., Molendi‐Coste, O., Cani, P. D., van Rooijen, N., Horsmans, Y., Leclercq, I. A. Kupffer cell depletion prevents but has no therapeutic effect on metabolic and inflammatory changes induced by a high‐fat diet. FASEB J. 25, 4301–4311 (2011). www.fasebj.org