Objective:To study the effect of transfection of antisense MBD1 gene eukaryotic expression vector on the expression of MBD1 gene in human cholangiocarcinoma cell line QBC939. Methods:The constructed antisense MBD1 gene eukaryotic expression vector was transfected into the human cholangiocarcinoma cell line QBC939 using lipofectamine transfection reagents, and positive cell clones were obtained using G418 selection after transfection. The constructed recombinant vector was transfected into QBC939 cells successfully and was confirmed by amplifying the exogenous neoR gene with PCR method. The expression level of MBD1 gene mRNA and protein was detected by RTPCR and FCM methods respectively. Results:Following the transfection, the MBD1 gene mRNA level in human cholangiocarcinoma cell line QBC939 decreased from 0.912±0.022 to 0.215±0.017, and the MBD1 gene protein level also decreased from (80.19±5.05)％ to (35.11±4.05)％. There were very significant differences on the expression both at the transcription and posttranscription levels of MBD1 gene between nontranfection group and the antisense MBD1 gene eukaryotic expression vector transfection group (P＜0.01). Conclusions:Transfection of the antisense MBD1 gene eukaryotic expression vector significantly reduced the expression level of MBD1 gene in human cholangiocarcinoma cell line QBC939, and suggestes that MBD1 gene plays an important role in the development of cholangiocarcinoma and that transfection of antisense MBD1 gene may be a new method to treat cholangiocarcinoma.