Abstract: Objective To construct a replication-deficient recombinant adenovirus carrying microRNA- 149(Ad-miR149) and to investigate its effect on the proliferation of human hepatocellular carcinoma cell lines. Methods The pre-microRNA-149 sequence was synthesized and inserted into multi-clone site in the adenoviral shuttle plasmid pDC315 in sense direction, and then the recombinant plasmid was co-transfected into human embryo kidney 293(HEK293) cells together with packaging plasmid pBHGlox(deltaE1,3). The recombinant adenovirus carrying hsa-microRNA-149 was selected and amplified. The effect of Ad-miR149 on the proliferation of human hepatocellular carcinoma cell lines was observed by MTT method. Results The replication-deficient recombinant adenovirus carrying microRNA-149 was packaged successfully. After virus amplification, the titer of Ad-miR149 was achieved to 5×109 pfu/ml. Virus replication was observed through the transmission electron microscope. Both adenovirus and microRNA-149 special fragment could be amplified from Ad-miR149 by PCR amplification. Compared with the negative control, only adenovirus special fragment could be amplified from Ad-LacZ. Ad-miR149 suppressed the proliferation of human hepatocellular carcinoma cell lines significantly detected by MTT. Conclusion The recombinant adenovirus carrying hsa-microRNA-149 is constructed successfully and its inhibiting effect on human hepatocellular carcinoma cell lines is confirmed preliminarily.