DHA (22:6n-3), an omega-3 fatty acid, is known for its anti-inflammatory properties. To enhance its bioavailability and therapeutic efficacy, lipid-core nanoformulations (LNC) have emerged as a promising delivery strategy. This strategy may offer therapeutic advantages in sepsis, a severe systemic inflammatory response to infection associated with high mortality. Herein, we developed, characterized, and tested a DHA nanoformulation in vitro (in flow-chamber assays) and in vivo (in TNF-α- stimulated and sepsis models). LNC _DHA was developed and characterized using laser diffraction and droplet Brownian motion techniques. LNC _DHA exhibited a homogeneous profile, with a mean diameter of 0.223 ± 0.00152 µm and a zeta potential of −12.83 mV. In CLP-induced sepsis, LNC _DHA limited bacterial dissemination, preserved body temperature, reduced leukocyte infiltration, and improved survival rates. Furthermore, LNC _DHA decreased pro-inflammatory cytokine levels in peritoneal exudate, plasma, and brain tissue, and elevated systemic and cerebral levels of resolvin D1 and D2. In vitro , LNC _DHA treatment reduced PSGL-1 and CD11a expression, increased rolling velocity, and decreased adhesion in murine neutrophils in flow chamber assays. In vivo , LNC _DHA reduced leukocyte rolling and adhesion while enhancing rolling velocity in TNF-ɑ-stimulated cremaster post-capillary venules assessed by intravital microscopy. LNC _DHA showed therapeutic potential in experimental sepsis by improving systemic and cerebral microcirculation, reducing inflammation, and increasing survival. This nanotechnology-based strategy represents a promising approach for treating inflammatory and infectious diseases.