Sargentodoxa cuneata is a tropical plant used in traditional Chinese medicine to treat intestinal inflammation. In this study, 3, 4-dihydroxyphenylethyl alcohol glycoside (DAG) was purified from the stem of S. cuneata using macroporous resins and its bioactivity was also investigated. The adsorption/desorption of DAG on macroporous resins was investigated systematically. HPD300 resin was selected as the most suitable medium for DAG purification. Further dynamic absorption/desorption experiments on the HPD300 column were conducted to obtain the optimal parameters. To obtain more than 95% DAG, a second stage procedure was developed to purify the DAG using SiliaSphere C18 with 8% v/v acetonitrile through elution at low pressure. Further investigation showed that DAG pretreatment significantly reversed the shortening of colon length, the increase in the disease activity index (DAI) scores and histological damage in the colon. Moreover, DAG greatly increased SOD and GPx activities, significantly decreased MPO and MDA activities and reduced the levels of pro-inflammatory cytokines in the colon. Free radical scavenging activities of DAG were assessed using DPPH, with an IC50 value of 17.03 ug/mL. Additionally, DAG suppressed ROS and proinflammatory cytokine production in LPS-stimulated RAW 264.7 macrophages by suppressing activation of the ERK1/2 and NF-κB pathways. The results were indicative of the antioxidant and anti-inflammatory properties of DAG. When viewed together, these findings indicated that DAG can be used to expand future pharmacological research and to potentially treat colitis.