Omega-3 polyunsaturated fatty acids (ω-3 FAs), including eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), exert neuroprotective and antioxidant effects. Their pleiotropic actions involve lipid mediators that regulate immune and inflammatory responses in both the systemic and central nervous system. We investigated the effects of early dietary supplementation with EPA and DHA on FAs composition in plasma and hippocampus. We also investigated inflammation, behavior and gut microbiota in spontaneously hypertensive rats (SHR). Male WKY and SHR rats received either a standard diet or ω-3 FAs (200 mg/kg/day) for 4 months (WKY+ω-3 and SHR+ω-3). We quantified fatty acid profiles and EPA/DHA-derived mediators, as well as Iba1 expression in the brain. SHR+ω-3 rats exhibited increased ω-3 FA levels in plasma and hippocampus, along with higher plasma levels of pro-resolving mediators in plasma. Exploratory behavior differed between SHR (treated and control) and WKY, suggesting hypertension-related alterations, although supplementation had no significant effect on behavior. SHR+ω-3 rats also showed a decrease in microglia cells in the hippocampus. The results revealed differences in bacterial families between groups, suggesting a modulation of the microbiota. We conclude that ω-3 FAs modified lipid profiles, induced changes in hippocampal microglia, and influenced gut microbiota on SHR rats, while the effects on behavior under these conditions remain inconclusive.