Our goal in this study is to improve the efficiency of docosahexaenoic acid (DHA) toward the enhancement of insulin signaling pathway in vivo via loading with ZnO NPs. To this end, two consecutive steps were undertaken, preparation of ZnO NPs by one-step solid-state reaction in dry conditions and calcinated followed by loading DHA. Both developed nanoparticles, with and without DHA were then characterized by TEM, SEM, EDX, and Zetasizer. For comparison between free and loaded DHA, four groups of rats were prepared to receive different treatments. Group I; healthy rats (reference), group II; diabetes (streptozotocin-induced), group III and group IV are diabetes orally administered with free DHA and DHA-loaded ZnO NPs (10 mg/kg bw/day), respectively. Blood samples were collected and analyzed where the results demonstrated that fasting blood sugar and insulin resistance were significantly increased in diabetic group along with upgrading in oxidative stress parameters emphasizing the oxidative properties of streptozotocin. HPLC analysis of cell membrane fatty acids resulted in the reduction of omega-6 and 9 and elevation of omega-3 after free DHA and DHA-loaded ZnO NPs streptozotocin treatments. DHA-loaded ZnO NPs had high performance in enhancing insulin signaling pathway as expressed in changes of phosphatidylinositol 3-kinase (PI3K) levels.