Abstract Acupuncture plays a vital anti-inflammatory action on sepsis through activating autonomic nerve anti-inflammatory pathways, such as sympathoadrenal medullary pathway, but the mechanism remains unclear. This study aims to explore the optimum parameter of electroacupuncture (EA) stimulation in regulating sympathoadrenal medullary pathway and evaluate EA’s anti-inflammatory effect on sepsis. To explore the optimum parameter of EA at homosegmental acupoint on adrenal sympathetic activity, the left adrenal sympathetic nerve firing rate evoked by different intensities of single shock electrical stimulation (ES) at ST25 in healthy male Sprague-Dawley (SD) rats were evaluated by in vivo electrophysiological recording, and the levels of norepinephrine (NE) and its metabolites were also examined using mass spectrometry. To verify the role of EA at ST25 in sepsis, the rat was given intraperitoneal injection lipopolysaccharide to induce sepsis model, and survival rate, clinical score, and the level of interleukin (IL)-6, IL-1β, and IL-10 were evaluated after EA application. We observed that 3 mA is the optimal intensity on activating adrenal sympathetic nerve, which significantly elevated the level of NE in the peripheral blood. For LPS-treated rats, EA at the ST25 apparently increased the survival rate and improved the clinical score compared to the control group. Furthermore, 3 mA EA at ST25 significantly decreased pro-inflammatory cytokines IL-6 and IL-1β and upregulated anti-inflammatory cytokine IL-10 compared to the Lipopolysaccharide (LPS)-treated group. Overall, these data suggest that 3 mA is the optimal EA intensity at ST25 to activate the sympathoadrenal medullary pathway and exert an anti-inflammatory effect on sepsis. Highlights 3 mA single shock ES at ST25 drove adrenal sympathetic nerve reflexs. 3 mA EA at ST25 upregulated NE concentration in peripheral blood. EA at ST25 improved the survival rate in a rat of LPS-induced sepsis model. EA at the ST25 upregulated anti-inflammatory IL10 and downregulated pro-inflammatory IL1β and IL-6 in LPS-treated rats.