Cholestatic liver disease (CLD) is characterized by disruptions in bile formation, secretion and excretion, leading to progressive liver injury, inflammation and fibrosis. Effective treatments to halt or reverse the progression of CLD remain limited. The Wnt/β-catenin signaling pathway has been implicated in the regulation of bile acid homeostasis and liver regeneration, playing a complex role in CLD pathophysiology. Tetrahedral framework nucleic acids (TFNAs), a class of anti-inflammatory and antioxidant DNA nanomaterials, have shown potential in promoting mammalian cell proliferation through activation of cell cycle and proliferation-related signaling pathways. However, their therapeutic potential in CLD has not been fully explored. In this study, we investigated the effects of TFNAs in an α-naphthyl isothiocyanate (ANIT)-induced mouse model of CLD. TFNAs demonstrated the ability to enter hepatocytes, where they activated the Wnt/β-catenin signaling pathway and enhanced ERK1/2 phosphorylation. These molecular changes resulted in significant improvements in liver injury markers, bile acid metabolism and liver regeneration. Complementary