Introduction: Intense local immune response, associated with recruitment and activation of lymphocytes and macrophages, and subsequent release of cytokines and other inflammatory mediators all cause tissue damage and contribute to many of the clinical features of colonic inflammation. The cholinergic anti-inflammatory pathway is a neuro-immune mechanism that regulates innate immune function and controls inflammation. The functional activity of this pathway can be modulated through its neuronal and non-neuronal (α7nACh receptors) cholinergic components. Objective: To explore the role of cholinergic anti-inflammatory pathway in acetic acid-induced acute colitis in rats. Materials and Methods: The colonic inflammation was induced in Sprague-Dawley rats (200-250 g; n=8/group) by intrarectal administration of 0.8 ml of 5% (v/v) acetic acid in 0.9% NaCl. The control rats received same volume of isotonic saline by the same route. The treatment groups were treated with either a natural acetylcholinesterase inhibitor huperzine A (HUP; 0.1 mg/kg) or a cholinergic agonist nicotine (NIC; 1 mg/kg) intraperitoneally. Drugs were given 5 min after induction of colitis and continued for 3 consecutive days. On the fourth day all rats were decapitated. The distal colon was scored and stored for the measurement of malondialdehyde (MDA) and glutathione (GSH) levels, and myeloperoxidase (MPO) activity. The presence of NFκB in colon was also examined. Formation of reactive oxygen species in colons was monitored by chemiluminescence (CL) method. Trunk blood was collected for the assessment of serum tumor necrosis factor (TNF-α) interleukin (IL)-1β and IL-10 levels. Results: The extent of colonic lesions in colitis group was reduced by HUP and NIC macroscopically (p<0.05), the microscopic score was only reduced by HUP treatment (p<0.05). Increased NFκB content in colitis group was reduced by HUP and NIC. Increase in colonic lipid peroxidation and lucigenin-enhanced CL in rats with colitis were attenuated by HUP (p<0.01). Stimulation of MPO activity in colitis group was attenuated by NIC (p<0.05). Increased serum TNF-α and IL-1β levels in colitis group were prevented by HUP (p<0.001 and p<0.05, respectively). Serum IL-10 level of the colitis and control groups was not significantly different but was increased by both NIC and HUP. Tissue GSH content did not differ among groups. Monitorization of body weigths during the course of study demonstrated a significant reduction in colitis rats which was reversed by NIC (p<0.05). Conclusion: Inhibition of AChE or treatment with a cholinergic agonist is beneficial on inflammatory parameters in a rat model of acute colitis. This supports the potential role of anti-inflammatory cholinergic pathway in the treatment of colonic inflammation.