Chemotherapy-induced oral mucositis (COM) is a common disorder but optimal treatment has not yet been established. COM is characterized by painful inflammation and ulceration involving PGE2. Hangeshashinto (TJ-14), a traditional Japanese herbal medicine (Kampo), has been prescribed for oral mucositis in Japan. Recently, a TJ-14 rinse solution (2.5 g/50 mL) was found to significantly reduce ulcer healing time in patients with grade 2 or worse COM in a randomized controlled trial. However, little is known regarding the anti-COM mechanism of TJ-14. The aim of this study was to identify the multiple actions of TJ-14 on the PGE2 system and cell migration by characterizing the action profiles of its main active ingredients, [6]-shogaol (6SG), wogonin (WGN), and berberine (BBR). COM was induced in hamsters by a combination of 5-fluorouracil (5-FU) administration and mild abrasion of the buccal mucosa. Hamsters were fed either a diet containing 2% (wt/wt) TJ-14 or a control diet throughout the experiments. Although buccal lesions were markedly aggravated by 5-FU, TJ-14 fed hamsters had significantly smaller lesions than animals on control diet and the ulcer healing time was also significantly reduced by half to a level comparable to that observed in humans. The amount of prostanoid synthesized by human oral keratinocytes (HOK) after stimulation with IL-1β for 6 h in the presence or absence of a test sample was measured by EIA. RT-PCR, phospho-MAPK, and COX-enzyme assays were performed to determine the mechanism of action. Levels of inducible PGE2 and PGF2α, metabolites of COX pathways, were reduced by TJ-14 (10-300 μg/mL) without inducing cytotoxicity, while LTB4 production was not affected by either IL-1β or TJ-14 addition. Gene expressions of COX-2, phospholipase A2, and PGE synthase were all down-regulated by TJ-14 exposure. Screening of the active ingredients in TJ-14 revealed that 6SG, WGN, and BBR significantly reduced PGE2 production. WGN and BBR inhibited induction of COX-2 mRNA probably through the blockage of c-Jun N-terminal kinase (JNK) and p38 phosphorylation. 6SG inhibited the enzymatic activity in PGE2 synthesis but did not decrease COX-2 mRNA expression, suggesting that 6SG might regulate PGE2 synthesis at the post-transcriptional level. Migration was evaluated after scratch wounding using sterile pipette tips. HOK migration was increased by TJ-14 (30-100 µg/mL) in a concentration-dependent manner. Pretreatment of HOK with IL-1β, which was highly expressed in inflamed sites of COM, resulted in decreased migratory activity. TJ-14 and BBR improved IL-1β-induced down-regulation of HOK migration. These data suggest that TJ-14 exhibits multiple actions against COM via PGE2 inhibition and promotion of wound healing. The main active ingredients, 6SG, WGN, and BBR, appear to exert different mechanistic actions.