Aflatoxin B₁ (AFB₁) is a prevalent mycotoxin contaminating food and environmental matrices that can induce hepatic injury, thereby posing a substantial threat to livestock production. Portulaca oleracea L. polysaccharide (POP) possesses diverse biological activities, including antioxidant and anti-inflammatory properties. However, the protective effects of POP against AFB₁-induced hepatic injury have not been fully elucidated. In the present study, a rabbit model of AFB₁-induced hepatic injury was established to investigate the hepatoprotective mechanisms of POP. Histopathological examination and liver function assessments demonstrated that POP markedly ameliorated hepatic histopathological alterations and decreased serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and alkaline phosphatase (ALP). Molecular analyses further demonstrated that POP attenuated AFB₁-induced oxidative stress and hepatocyte apoptosis, effects that were associated with activation of the PI3K/AKT signaling pathway and suppression of the mitochondria-mediated apoptotic pathway. Furthermore, microbiome profiling and targeted short-chain fatty acid (SCFA) metabolomics analyses indicated that POP supplementation contributed to the preservation of intestinal barrier integrity and intestinal microbiota stability, concurrent with the attenuation of AFB₁-induced intestinal and hepatic injury and elevated SCFA production by intestinal microbes. Overall, POP exerts prominent preventive protective effects against AFB₁-induced liver injury, which may be closely associated with alterations in intestinal microbiota composition and microbial short-chain fatty acid metabolism, as well as the activation of the PI3K/AKT signaling pathway. These findings provide novel insights into the mechanisms underlying AFB₁-induced hepatic injury.