To explore the effects of Mucins (MUC)1-shRNA on the proliferation and hypoxia inducible factor (HIF)-1alpha expression of human cholangiocarcinoma (CCA) QBC939 cells in vitro. MUC1-shRNA was constructed and transfected with Lipofectamine™ 2000 into cultured CCA cells. MUC1 mRNA and protein expression levels were determined by RT-PCR and Western blot, respectively. The cellular proliferation and HIF-1alpha expression of QBC939 cells were evaluated by the MTT assay and Western blot, respectively. After transfection, the expression levels of MUC1 mRNA and protein in the experimental group decreased significantly in QBC939 (P < 0.01). The proliferation of MUC1 shRNA-transfected group was 0.30 ± 0.05, 38.32 ± 1.43%, 15.18 ± 1.32%, and there were remarkable differences when compared with the control groups (P < 0.05). Significant inhibition of HIF-1alpha protein expression in MUC1 shRNA-transfected group was also discovered (P < 0.05). MUC1-shRNA could inhibit proliferation and significantly weaken HIF-1alpha protein expression of QBC939 cells, suggesting its potential as a therapeutic target of CCA.