The paper was aimed to detect the expression of Skp2, p27(kip1) (p27), and p-p27 (Thr187) in patients with cholangiocarcinoma (CCA). Western blot and immunohistochemistry were used to analyze the expression and subcellular localization of p27, Skp2, and p-p27 (Thr187) in tissue specimens of 53 patients with CCA and 10 with chronic proliferative cholangitis (CPC). Besides, the relationships of p27, Skp2, and p-p27 (Thr187) with clinicopathologic findings were examined, followed by analysis of the relationships of p27 expression with p-p27 (Thr187) and Skp2 in CCA tissue. The expression of p27 was lower in CCA than CPC tissue, and the expression of Skp2 and p-p27 (Thr187) was higher in CCA than CPC tissue (P < 0.05). The positive p27 and Skp2 proteins were mainly expressed in nucleus and cytoplasm of CCA, and p-p27 (Thr187) was only observed in nucleus. The expression of p27, Skp2, and p-p27 (Thr187) was closely associated with tumor grade and TNM stage (P < 0.05). A significantly negative correlation between the expression of p27 and Skp2 (r = -0.480, P < 0.05) and between the expression of p27 and p-p27 (Thr187) (r = -0.387, P < 0.05) was observed. Skp2-dependent proteasomal degradation of p27 plays role in the malignant transformation of CCA. Besides, the expression of p27, Skp2, and p-p27 (Thr187) may serve as markers for the progression of CCA.