In the ayurvedic system of medicine polyherbal formulations were frequently used to enhance the activity or counteract the toxic effect of compounds, from other plants but may also act synergistically with other constituents from the same plants. Therefore, the purpose of the study was to formulate a polyherbal formulation and investigate its anti-ulcer effect on various ulcer models such as pylorus ligation, indomethacin, cold restraint stress, HCl/Ethanol and cysteamine induced ulcers in animals. The Polyherbal formulation (PHF) consists of seven medicinal plants namely Aegle marmelos, Elettaria cardamomum, Glycyrrhiza glabra, Citrus aurantifolia, Rosa damascena, Cissus quadrangularis and Saccharum officinarum. Based on acute toxicity study, the PHF was considered as safe and 3 dose (100, 200 and 400 mg/kg) levels were used. Oral administration of PHF, once daily for 3 days showed dose-dependent anti-ulcer activity. The PHF exhibited significant (P<0.001) decrease in ulcer index and enhance the % protection of ulcer at all the 3 doses. Further study on gastric mucosal factors showed that it significantly decreased the offensive factors like acid (control: 214.08±5.65, PHF: 109.65±4.42) and pepsin (control: 49.35±1.35 and PBF: 20.00±0.92). PHF increased the defensive factors like mucin secretion (TC:P ratio of control: 1.55±0.06 and PHF: 4.24±0.07) and life span of mucosal cells (DNA content of gastric juice in control: 228.80±3.12 and PHF: 119.82±6.57). PHF showed significant antioxidant effect in stressed animals (Stress control: LPO, SOD and CAT were 242.92±7.99, 149.70±3. 10 and 20.40±1.58 respectively; PHF reduced the LPO and SOD to 157.92±7.26 and 117.88±4.42 respectively and increase in mucosal CAT to 27.18+1.91). PHF did not have any effect on cell proliferation in terms of DNA mg/mg protein or glandular weight. The hexosamine (Stress control 20.48±1.22: PHF 100, 200and 400mg/kg were 28.17±0.71, 32.35±1.18 and 36.13±0.43 respectively) and gastric wall mucus (Stress control 812.47±41.54: PHF 100, 200and 400mg/kg were 997.82±14.32, 1042.5±20.73 and 1196.8±13.17 respectively) was significantly (P<0.001) increased by the PHF. From the results, it was concluded that PHF had significant anti-ulcer effects and this might be due to its effects on both offensive and defensive mucosal factors as well as scavenging of free radicals generation. © 2008 Elsevier B.V., All rights reserved.