Fisetin, quercectin, and myricetin are flavonols with similar structure but different number of hydroxyl groups. The present research focused on the anti-inflammatory activities and the underlying mechanisms of fisetin, quercetin, and myricetin. In vitro experiment showed that the number of hydroxyl group in flavonols obviously affects their anti-inflammation activity in lipopolysaccharide-stimulated RAW264.7 cells. Among flavonols in safe concentrations, they suppressed the overproduction of nitric oxide, and fisetin showed the best activity with an inhibition rate of 52% at 20 μM. These flavonols reduced the expression levels of ROS, TNF-α, and IL-6 with variable effects. And they could block the activation of NF-κB and MAPK pathways by partially suppressing levels of phosphorylation of IκBα, p65, JNK, ERK, MEK, and reducing the nuclear translocation of NF-κB p65. In addition, several degradation products of fisetin were formed by methylation and glucuronidation. And quercetin and myricetin were transformed into several new products, including methylated quercetin and methylated myricetin, respectively. According to the comparative result, fisetin is an excellent anti-inflammatory reagent. Therefore, in vivo research was conducted to test whether fisetin has the liver protective and anti-inflammatory effects in D-GalN/LPS-induced acute liver injury from C57BL/6 mice. Results indicated that the pretreatment of fisetin could suppress the levels of AST, ALT, IL-1β, IL-6, and TNF-α in serum and reduced the MDA and CAT levels in hepatic tissues. In macroscopic view and H&E staining analysis, fisetin pretreatment reduced hepatic congestion, liver necrosis, and inflammatory cell infiltration. The underlying mechanism of this hepatoprotective effect was possibly associated with NF-κB signaling pathway. Thus, fisetin, quercetin, and myricetin are three potential agents again inflammation and among which fisetin showed the excellent effect. Fisetin may be a promising agent for the treatment of acute liver injury.