In our previous study, preliminary data indicates that Poria cocos polysaccharides (PCP) shows beneficial hepatoprotection against acetaminophen (APAP)-induced liver injury in mice. However, biological molecular mechanism warrants to be further discussed. In current study, a number of biochemical tests and immunoassays were subjected to respective PCP-dosed mice in vivo and liver cells in vitro. As a result, PCP-treated mice showed reduced contents of inflammatory cytokines (tumor necrosis factor [TNF]-β and TNFsR-I), enzymological molecules (alanine aminotransferase, aspartate aminotransferase, and lactate dehydrogenase [LDL]), and heat shock protein 90 (Hsp90) after APAP exposure. Additionally, immunostaining assays exhibited that lowered-positive cells of cleaved-caspase-3, cleaved-poly ADP ribose polymerase, and Hsp90-labeled cells in PCP-treated livers were observed, and increased cluster of differentiation 29 (CD29), CD73-positive cells in the spleen were detected. Further, PCP-treated mouse liver cells resulted in increased cell growth, reduced LDL level. Increased proliferating cell nuclear antigen (PCNA), P38 mitogen-activated protein kinase (MAPK)-labeled cells and decreased Hsp90-positive cells in APAP-exposed liver cells were observed dose-dependently after PCP cotreatments. Collectively, our present experimental findings elucidate that PCP beneficially play hepatoprotective effects against APAP-lesioned liver cells in vivo and in vitro, potentially through the molecular mechanisms of suppressing cell death, reducing hepatocellular inflammatory stress and Hsp90 bioactivity.