Pedunculoside (PE), a triterpenoid saponin isolated from Ilex rotunda Thunb., exhibits an anti-inflammatory effect; however, its hepatoprotective effects and pharmacokinetics are still unclear. Thus, an integrated strategy using network pharmacology and pharmacokinetics was used to investigate the hepatoprotective effects of PE. A simple, sensitive, and reliable LC-MS/MS method was established for the quantitative analysis of PE to elucidate its pharmacokinetics in normal rats and those with acute liver injury (ALI). LO2 cells and carbon tetrachloride–treated rats were used to evaluate the hepatoprotective effects and mechanism of PE in vitro and in vivo, respectively. The blood concentration curve demonstrated a decrease in clearance (CL) and an increase in the area under the curve (AUC(0-∞)) and mean residence time (MRT) in rats with ALI. Normal rats in the intravenous injection group had a CL of 36.80 L/h/kg, AUC(0-∞) of 1092.01 μg/L•h, and MRT(0-∞) of 0.93 h. In contrast, the CL of rats with ALI was 34.54 L/h/kg, AUC(0-∞) was 1170.01 μg/L•h, and MRT was 1.16 h. Similar results were obtained for rats in the intraperitoneal injection group. Qualitative studies of rat metabolites identified 19 metabolites, indicating that PE mainly undergoes phase I metabolism followed by phase II metabolism to a lesser extent. As rats with ALI showed a liver-targeting trend, network pharmacology was used to predict 20 potential ALI-related targets, among which the nuclear factor-kappa B (NF-κB) pathway had the strongest correlation. PE exhibited a protective effect on ALI by decreasing the levels of alanine transaminase, aspartate transaminase, and the pro-inflammatory cytokines tumor necrosis factor-α, interleukin (IL)-1β, and IL-6, wherein the mechanism was related to the NF-κB pathway. Thus, PE exhibited a significant protective effect against ALI through the NF-κB pathway.