Acute liver injury is characterized by fibrosis, inflammation, and apoptosis, leading to liver dysfunction, cirrhosis, or cancer and affecting the clinical outcome in the long term. However, no effective therapeutic strategy is currently available to treat the disease. Cimicifuga heracleifolia Kom., a commonly used wild vegetable, has been reported to have multiple biological functions. However, the pharmacological function of Cimicifuga polysaccharides is still unclear. In this study, a neutral polysaccharide (CHP-N-1) from Cimicifuga heracleifolia was characterized, and its protective effects against CCl4-induced HepG2 cell and liver injury were investigated. Based on the data from High-Performance Liquid Chromatography, Gas Chromatography-Mass Spectrometer and Nuclear Magnetic Resonance Spectroscopy, it was determined that CHP-N-1 consisted of amylose, glucomannan, and arabinoxyloglucan. Amylose was composed of α-glucose linked by α-1,4-glycosidic bonds. Glucomannan was primarily comprised of β-1,4-d-mannopyranosyl and β-1,4-d-glucopyranosyl residues without any branches. The backbone of arabinoxyloglucan was composed of (1 → 4)-linked β-d-glucopyranosyl units. Some glucopyranosyl residues in the backbone were substituted mainly at C-6 by the side chain of β-d-Xylp(1→, and α-Araf-(1 → 5)-α-L-Araf(1 → . In vitro experiments revealed that CHP-N-1 exhibited protective effects against CCl4-induced damage in HepG2 cells. In vivo experiments demonstrated that CHP-N-1 exhibited a hepatoprotective effect by enhancing antioxidant enzyme activity, inhibiting lipid peroxidation, and reducing the activity of pro-inflammatory mediators. Besides, CHP-N-1 could attenuate oxidative stress and inflammatory responses by activating the TLR4-mediated NF-κB signaling pathways. These findings demonstrated that CHP-N-1 may serve as a supplement for alleviating chemical liver damage.