We examined whether a low amount of dietary long-chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) modulated phosphatidylinositol 3'-kinase (PI 3-kinase) activity and downstream Akt phosphorylation differently in normal or insulin-resistant rats. Rats were fed for 28 days with either a control diet containing 14.6% of metabolizable energy (ME) as peanut-rape oil (PR) or an n-3 diet where 4.9% of ME as PR was replaced by fish oil. Over the last 5 days, rats received 9 per thousand NaCl or dexamethasone (1 mg/kg). Insulin stimulation of both PI 3-kinase activity and Akt serine(473) phosphorylation and modulation of GLUT4 content were studied in liver, muscle, and adipose tissue (AT). Glucose tolerance and insulin sensitivity were determined by an oral glucose challenge. In muscle and AT, LC n-3 PUFA abolished insulin-stimulated PI 3-kinase activity. These effects were not paralleled by defects in Akt serine(473) phosphorylation, which was even increased in AT. Dexamethasone abolished insulin-stimulated PI 3-kinase activity in all tissues, whereas Akt serine(473) phosphorylation was markedly reduced in muscle but unaltered in liver and AT. Such tissue-specific dissociating effects of LC n-3 PUFA on PI 3-kinase/Akt activation took place without alteration of glucose metabolism. Maintenance of a normal glucose metabolism by the n-3 diet despite abolition of PI 3-kinase activation was likely explained by a compensatory downstream Akt serine(473) phosphorylation. The inability of LC n-3 PUFA to prevent insulin resistance by dexamethasone could result from the lack of such a dissociation.