Acetaminophen (APAP) overdose has become a leading cause of acute liver failure (ALF) in the United States and Europe. Alismatis rhizoma, the dried rhizome of Alisma orientale (Sam.) Juzep. or Alisma plantago-aquatica Lin, is a traditional medicinal herb. Our previous studies demonstrated that refined polysaccharides derived from Alismatis rhizoma exerted protective effects against APAP-induced liver injury. Nevertheless, the structures, biological activities, and structure-activity relationships of its glycans remained to be elucidated. In the present study, four novel glycans, designated ARP70-1, ARP70-2, ARP70-3, and ARP70-4, were isolated from Alismatis rhizoma. These polysaccharides were primarily composed of arabinose and galactose, with the predominant moieties identified as α-Araf-(1→, →5)-α-Araf-(1→, →3)-β-Galp-(1→, →6)-β-Galp-(1 → and →4,6)-β-Galp-(1→. In vitro experiments demonstrated that arabinogalactans mitigated APAP-induced injury in HepG2 cells by enhancing the levels of GSH, SOD, and CAT, while reducing MDA production. In vivo pharmacological evaluation of ARP70-1 using an APAP-induced acute liver injury mouse model showed that ARP70-1 enhanced hepatic antioxidant capacity, reduced inflammatory cytokine levels, and increased the abundance of beneficial intestinal bacteria, including Muribaculum, Prevotellaceae_UCG_001, Bacteroides, Parabacteroides, and Ruminococcus_torques_group. Additionally, ARP70-1 elevated intestinal levels of short-chain fatty acids (SCFAs) such as acetate, propionate, isobutyrate, 2-methylbutyrate, and isovalerate. Moreover, ARP70-1 promoted the hepatic expression of genes involved in bile acid metabolism and transport (CYP7A1, SLC10A1, ABCB11, ABCC3, BAAT, SULT2A1, and SULT2A8), and increased the levels of conjugated bile acids, including tauro-ursodeoxycholic acid (TUDCA), tauro-chenodeoxycholic acid (TCDCA), tauro-ω-muricholic acid (TωMCA), tauro-hyodeoxycholic acid (THDCA), and tauro-deoxycholic acid (TDCA). These effects collectively improved bile acid synthesis, transport, metabolism, and excretion, alleviated APAP-induced cholestasis, and conferred hepatoprotective effects.