Background Smad4 is found mutated in many cancers. It acts as a tumor suppressor in the regulation of TGF-8 signaling pathway. The objective of this work was to study the expression of Smad4 in intrahepatic cholangiocarcinonna (ICC) and its relationship with the biological behavior and prognosis of the disease. Methods Forty-nine paraffin-embedded ICC specimens and nine normal liver tissues were analyzed by imnnunohistochemical methods using Smad4 monoclonal antibodies. The expression of Smad4 was compared with the clinical pathological characteristics of the patients. Results The expression of Smad4 was 100% positive in normal liver tissues, which was higher than that in the ICC (44.9%). Negative labeling of the Smad4 protein was found in 26.1% (6/23) of well-differentiated ICCs and 61.5% (16/26) of poorly to moderately differentiated ICCs, and 34.3% (12/35) and 71.4% (10/14) showed negative Smad4 labeling (P=0.018) of ICC at pathological Tumor Node Metastasis (pTNM) stage I-II and pTNM stage III-IV separately. Furthermore, 72% (8/11) of lymph node metastatic ICCs and 73.3% (11/15) of intrahepatic metastatic ICCs showed negative labeling of the Smad4 protein. The loss of Smad4 expression in those metastatic ICCs was significantly more severe compared with non-metastatic ICCs (P=0.000). Conclusions The expression of Smad4 was associated with the histological grade, clinical stage, and metastasis of ICC (P < 0.05). The detection of Smad4 may be helpful in determining the degree of malignancy and prognosis of ICC.