We aimed to define changes in membrane fatty acids and signaling proteins induced by virgin olive oil (VOO) consumption in elderly persons with type 2 diabetes (n = 16) compared to a control group (n = 28). The fatty acid composition was determined by gas chromatography and G-protein subunits and protein kinase C alpha (PKCα) by immunoblotting. VOO consumption increased the monounsaturated fatty acid content in phospholipids and cholesterol esters in both groups. In contrast, saturated fatty acids were decreased only in phospholipids. The levels of Gαo, Gβ, and PKCα were significantly lower in diabetics than in controls. However, whereas VOO consumption reduced Gαs, Gβ, and PKCα in both groups, reduction in Gαi was observed only in diabetics. These results indicate that long-term VOO consumption modifies the fatty acid composition of plasma membrane, which influences the association of G proteins and PKCα with the lipid bilayer. These combined effects probably account for the positive effects of VOO on glycemic homeostasis.