Background: Pair medicine is a unique feature of Chinese medicine. It refers to a specific composition of the two drugs usually used in the formulation of composite medicine, which could increase synergistically or decrease the side effects of one medication. Atractylodis macrocephalae Rhizoma (AMR), a generally used drug, possessed the spleen-tonifying action always used with Rhubarb as a pair medicine to decrease the side effect of Rhubarb. Aim: To investigate the effect of Atractylodis macrocephalae Rhizoma (AMR) and its effective constituents to alleviate the Rhubarb's side effect in their pair medicine. Materials and Methods: The mice were administered Rhubarb until the induction of diarrhea followed by gastrointestinal injury. The gastrointestinal injured mice were treated with AMR's water decoction and its subfractions for 5 consecutive days. The decoction and subfraction were chemically characterized using the High-performance liquid chromatography (HPLC) with a diode-array detector (DAD) analysis. Results: The results showed that AMR's water decoction (6 g/kg) was discovered to be the most effective dose to treat gastrointestinal injury induced by Rhubarb. Body weight, thymus and spleen indexes, the intestinal propulsion rate, and D-xylose absorption in mice with diarrhea and intestinal injury were analyzed to reveal the significant difference between the tested and control groups (P < 0.01). The Water Eluated Fraction (WEF), Petroleum Ether Fraction (PEF), and Crude Polysaccharide Fraction (CPF) could not only increase the levels of amylase, gastrin, and vasoactive intestinal peptide significantly but also ease diarrhea and intestinal injury situation compared with the control group (P < 0.01). Conclusion: AMR and its subfractions effectively ameliorate gastrointestinal side effects of Rhubarb and its components. WEF mainly contained 5-hydroxymethyl furfural and small molecular sugar. PEF mainly composed of sesquiterpene lactone-atractylenolides, while CPF mainly composed of inulin-type oligosaccharides elucidated by HPLC analysis, was the most effective subfraction to alleviate diarrhoea and gastrointestinal injury induced by Rhubarb.