Acupuncture plays a vital anti-inflammatory action in sepsis by 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 the sympathoadrenal medullary pathway and evaluate EA's anti-inflammatory effect on sepsis. To determine the optimum parameter of EA at homotopic 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 rats were evaluated by in vivo electrophysiological recording, and the levels of norepinephrine (NE) and its metabolites normetanephrine (NMN) were also examined using mass spectrometry. To verify the role of EA at ST25 in sepsis, the rats were given an intraperitoneal injection of lipopolysaccharide (LPS) 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 for 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 LPS-treated group. Overall, our data suggested that 3 mA is the optimal EA intensity at ST25 to activate the sympathoadrenal medullary pathway and exert an anti-inflammatory effect in sepsis.