BACKGROUND Anoectochilus roxburghii (Orchidaceae) (AR) has been widely used to treat liver injury in China, but its underlying mechanisms remain elusive. Network pharmacology was utilized to assess the hepatoprotective effects of quercetin (Que)-containing AR, and to validate the anti-liver injury effects of Que in a mouse model of liver injury. MATERIAL AND METHODS Network pharmacology analysis was performed to determine bio-active compounds in AR. The core therapeutic targets of AR against liver injury were identified using a protein-protein interaction network. Biological function and pathway enrichment were analyzed based on the identified core therapeutic targets. The hepatoprotective effects of Que in a mouse model of liver injury induced by CCl4 were assessed to verify the reliability of network pharmacology analysis. RESULTS Seven bio-active compounds of AR met drug screening criteria and 17 core therapeutic targets of AR against liver injury were identified. Biological function analysis demonstrated that the therapeutic effects of AR against liver injury were chiefly associated with the suppression of inflammation and immunity; and pathway enrichment analysis showed that nuclear factor-kappa B (NF-kappaB) and tumor necrosis factor (TNF) signaling pathways were associated with the inflammatory responses. Experimental validation in a mouse model showed that AR exerted anti-inflammatory effects by regulating the NF-kappaB signaling pathway, a finding that also confirmed the reliability of network pharmacology analysis. CONCLUSIONS The bio-active compounds identified in AR and the elucidation of their mechanisms of action against liver injury provide a theoretical basis for designing agents that can prevent or suppress liver injury.