The effect of the total saponins from Rosa laevigata Michx fruit (RLTS) against acetaminophen (APAP)-induced liver damage in mice was evaluated in the present paper. The results showed that RLTS markedly improved the levels of liver SOD, CAT, GSH, GSH-Px, MDA, NO and iNOS, and the activities of serum ALT and AST caused by APAP. Further research confirmed that RLTS prevented fragmentation of DNA and mitochondrial ultrastructural alterations based on TdT-mediated dUTP nick end labeling (TUNEL) and transmission electron microscopy (TEM) assays. In addition, RLTS decreased the gene or protein expressions of cytochrome P450 (CYP2E1), pro-inflammatory mediators (IL-1β, IL-4, IL-6, TNF-α, iNOS, Bax, HMGB-1 and COX-2), pro-inflammatory transcription factors (NF-κB and AP-1), pro-apoptotic proteins (cytochrome C, p53, caspase-3, caspase-9, p-JNK, p-p38 and p-ERK), and increased the protein expressions of Bcl-2 and Bcl-xL. Moreover, the gene expression of IL-10, and the proteins including LC3, Beclin-1 and Atg5 induced by APAP were even more augmented by the extract. These results demonstrate that RLTS has hepatoprotective effects through antioxidative action, induction of autophagy, and suppression of inflammation and apoptosis, and could be developed as a potential candidate to treat APAP-induced liver damage in the future.