Extracellular high mobility group box 1 (HMGB1) is a lethal pro-inflammatory mediator in endotoxin shock. Hyperacetylation of HMGB1, regulated by histone acetyltransferases (HATs) and histone deacetylases (HDACs), changes its subcellular localization and secretion to the extracellular matrix. Paeonol (2'-hydroxy-4'-methoxyacetophenone), one of the main active components of Paeonia suffruticosa, exerts anti-inflammatory effects. Our previous study demonstrated that Paeonol inhibited the relocation and secretion of HMGB1 in lipopolysaccharide (LPS)-activated RAW264.7 cells. However, it is still unclear whether Paeonol can regulate HATs/HDACs, which are responsible for the translocation of HMGB1 from nucleus to cytoplasm. To answer this question, P300 (a transcriptional coactivator with HATs) and HDAC3 were investigated using RT-qPCR and western blotting. The results showed that HMGB1 translocated from the nucleus to the cytoplasm, accompanied by upregulation of P300 and downregulation of HDAC3 in LPS-induced RAW264.7 cells. Paeonol, however, reversed the expression of P300 and HDAC3 significantly, suggesting that Paeonol may be involved in the acetylation of HMGB1 by regulating P300/HDAC3. Then, the effect of HDAC3 on the nucleocytoplasmic transportation of HMGB1 by HDAC3-SiRNA was evaluated. The results demonstrated that the inhibition of HDAC3 resulted in the nucleocytoplasmic translocation of HMGB1, with or without LPS stimulation. Moreover, Paeonol had no effect on the translocation of HMGB1 following ablation of HDAC3. These findings support the hypothesis that Paeonol can inhibit the translocation and secretion of HMGB1 in LPS-induced RAW264.7 cells by upregulating the expression of HDAC3. Paeonol may therefore be a valuable candidate as an HMGB1-targeting drug for inflammatory diseases via upregulation of HDAC3.