Abstract Purpose Recently, cholinergic anti-inflammatory pathway manipulation has been proposed as a new strategy to control cytokine production in sepsis. We investigated whether hypercytokinemia can be controlled via this pathway in an animal model of sepsis, with concomitant monitoring of autonomic nervous activity involving heart rate variability (HRV) analysis of electrocardiographic R-R intervals. Methods Sixty-eight adult male Sprague–Dawley rats were used (28 for examination of cytokine production and autonomic nervous activity; 40 for survival analysis). Each part of the study involved four animal groups, including two control groups without drug administration. Sepsis was induced by cecal ligation and puncture (CLP). Distigmine bromide, a peripheral, non-selective cholinesterase inhibitor (0.01 mg/kg), was administered subcutaneously 90 min after surgery. Continuous electrocardiograms were recorded for 5 min before and after surgery (at intervals of 5 h) in CLP and sham-operated animals for HRV analysis. Blood samples were collected 20 h after surgery for serum cytokine and catecholamine assay. Results On HRV analysis, distigmine inhibited reduction of total power and high-frequency components in CLP animals ( P < 0.05). Distigmine significantly inhibited cytokine induction (IL-6 and IL-10) ( P < 0.01) as well as increase in serum levels of noradrenaline and dopamine ( P < 0.05). Distigmine did not significantly improve CLP animal survival rate. Conclusions The cholinesterase inhibitor distigmine inhibited induction of inflammatory cytokines and catecholamines as well as HRV suppression in a rat CLP model, suggesting that an agent modulating the cholinergic anti-inflammatory pathway can control excess cytokine production involved in the pathogenesis of severe sepsis/septic shock.