Acute liver injury (ALI) leads to inflammatory response and tissue damage. Inflammatory activation of infiltrative macrophages plays a critical role in liver histology destruction and dysfunction. Hydroxytyrosol (3,4-dihydroxyphenil-ethanol, HT), one of the polyphenols extracted from extra virgin olive oil, currently acts as a treatment for neuroinflammatory responses, but its effect on ALI is elusive. The present study aims to examine the mechanism of HT in macrophages inflammation and evaluate treatment effect of HT on ALI. In vitro, the expressions of type M1/M2 macrophages biomarkers (CD11c/CD206) and cytokines (TNF-α, IL-1β, IL-6, IL-10, IL-4) following lipopolysaccharide (LPS) stimulation and HT administration were detected using immunofluorescence, quantitative real-time polymerase chain reaction (qRT-PCR), and enzyme-linked immunosorbent assay (ELISA). Mechanically, HT was used to treat cells and phosphorylation level of extracellular-signal-regulated kinase 1/2 (ERK1/2) protein in cells was analyzed using Western blotting. In murine acute liver injury, inflammatory cytokines and liver injury degree were exhibited by qRT-PCR, IHC and HE staining. Furthermore, hepatic function was exhibited via hepatic metabolic enzymes (ALT/AST) and total bilirubin (TBil) in serum. It was demonstrated that HT treatment attenuated M1 macrophages and increased M2 macrophages after LPS stimulation. Furthermore, the pro-inflammatory cytokine level was descended, while an-inflammatory cytokine was increased via HT suppressing ERK pathway in macrophages. In vivo, HT reduced inflammatory level and mitigated hepatic histological injury, thus ameliorating liver function after acute liver injury. HT exerts a hepatoprotective and anti-inflammation effect on acute liver injury, which restrains inflammation by inhibiting ERK pathway and regulating macrophages polarization. Moreover, HT prevents liver tissues from inflammatory injury. Therefore, HT serves as a potential implication to treat ALI through modulating inflammation of macrophages.