We examined if a low amount of dietary long chain n-3 polyunsaturated fatty acids (LC n-3 PUFA) modulated differently phosphatidylinositol 3'-kinase (PI 3-kinase) activity and downstream Akt phosphorylation, in normal or insulin-resistant rats. Rats were fed 28 days with either a control diet containing 14.6 % peanut-rape oil (PR) or a n-3 diet where 4.9% of PR were replaced by fish oil. Over the last 5 days, rats received saline or dexamethasone (1 mg/kg). Insulin stimulation of both PI 3-kinase activity and Akt serine⁴⁷³ phosphorylation, and modulation of GLUT 4 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 serine⁴⁷³ phosphorylation which was even increased in AT. Dexamethasone abolished insulin-stimulated PI 3-kinase activity in all tissues while Akt serine⁴⁷³ 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 in spite of abolition of PI 3-kinase activation was likely explained by a compensatory downstream Akt serine⁴⁷³ phosphorylation. The inability of LC n-3 PUFA to prevent insulin resistance by dexamethasone could result from the lack of such a dissociation.