In the current study, we demonstrated that AMDHD1 interacted with SMAD4 and SMAD2/3, which activated Transforming Growth Factor β (TGF-β) signaling pathway and subsequently suppressed the malignant traits of CCA cells, revealing a novel aspect of AMDHD1’s role in cancer biology and offering potential new avenues for therapeutic intervention. The gene discussed is SMAD4; the disease is cholangiocarcinoma.