In Turkish folk medicine, aerial parts of Ajuga chamaepitys (L.) Schreber subsp. chia (Schreber) are used for the treatment of diarrhea. The crude methanolic extract of aerial parts of A. chamaepitys subsp. chia was sequentially fractionated into five subextracts; n-hexane, dichloromethane, ethyl acetate, n-butanol and aqueous extracts. Effects of the methanol extract, subextracts and fractions were investigated in acetic acid–induced rat colitis model. The MeOH extract and n-BuOH subextract have regulated the caspase-3, myeloperoxidase, TNF-α, IL-6 levels and antioxidant parameters. After confirmation of the activity against ulcerative colitis, n-BuOH subextract was subjected to more chromatographic separation for the isolation of compounds ajugoside (1), asperulosidic acid (2) and deacetyl-asperulosidic acid (3). As a conclusion, A. chamaepitys subsp. chia can be used in cell, tissue, or individual-specific treatments that will be developed in the future treatment of IBD, or as a complementary therapeutic agent that contributes to these treatments.