Liver injuries caused by various industrial chemicals represent a serious health concern worldwide. Flexirubins are a novel class of naturally occurring bacterial pigments whose bioactivity remains largely unexplored. The present study evaluated the hepatoprotective effects of flexirubin pigment extracted from the bacterium Chryseobacterium artocarpi against CCl4-induced acute liver injury in mice. Flexirubin was applied at three different oral doses, 125, 250 and 500 mg/kg bw/d for seven consecutive days. Treatment of animals with flexirubin before exposure to CCl4 (10 mL/kg bw dissolved in olive oil, 1:1 v/v) significantly decreased the elevated serum levels of ALT, AST, ALP, LDH and TBL. Flexirubin pretreatment showed a great capability for attenuating the CCl4-induced oxidative stress by decreasing the level of liver MDA, and increasing the antioxidant enzyme activities of liver SOD and CAT, and the levels of GSH and TAC. Flexirubin also alleviated the histopathological alterations in liver by prohibiting steatosis, ballooning degeneration, leukocytic infiltration and necrosis. Immunohistochemical analysis demonstrated that flexirubin has a significant anti-apoptotic activity against CCl4 via upregulation of Bcl-2, and downregulation of Bax, Caspase-3 and TGF-β1. Flexirubin also exhibited a remarkable anti-inflammatory activity against CCl4 through its suppressive action on TNF-α, COX-2 and CD-45. Flexirubin could trigger upregulation of the Nrf2/HO-1 signaling pathway mediating protection against CCl4. In silico molecular docking revealed flexirubin as a potential inhibitor against two target proteins, TGF-β1 and TACE. The results proved the effectiveness of flexirubin as a significant source of natural compounds for its use in drug formulation strategies to offer protection against hepatotoxins. Copyright © 2022. Published by Elsevier Inc.