<p>Hwangkeumjakyak-tang (HJT), a Korean traditional herbal medicine, is known to clear phlegm, relieve congestion, and reduce inflammation. However, there is a lack of research on the efficacy of HJT in other diseases. The present study aimed to investigate whether HJT could protect against liver injury. We first examined the effects of HIT in vitro by assessing its toxicity and the phosphorylation and nuclear translocation of nuclear factor erythroid 2-related factor 2 (Nrf2) in human HepG2 cells. Additionally, we investigated whether HJT inhibited oxidative stress by measuring reactive oxygen species (ROS) production and intracellular levels of reduced glutathione (GSH). We further assessed the effects of HJT in vivo by analyzing liver damage parameters using an acetaminophen (APAP)-induced liver damage mouse model. Treatment with HJT significantly reduced the elevated serum levels of ALT and AST and the cytokine release induced by APAP injection. Furthermore, we verified that treatment with HJT suppressed APAP-induced ROS production and GSH depletion in the mouse liver, suggesting that HJT inhibited APAP-induced liver injury by blocking oxidative stress in vivo. To identify the potential relationship between the liver and intestine for liver disease therapy, we also performed 16S rRNA amplicon sequencing to analyze the gut microbiome and confirmed that HJT regulated APAP-induced changes in the gut microbiome pattern. In conclusion, HJT, a Nrf2 activation inducer, exhibits a cytoprotective effect on APAP-induced liver injury and can regulate the gut microbiome altered by the administration of APAP.</p>