Objective To analyze the antitumor effects of microRNA-144 (miRNA-144) gene overexpression and corresponding changes in intestinal flora in a mouse model of hepatoma. Methods First, we transplanted ascites hepatoma cells in mice. To observe the general effects of overexpression of the miRNA-144 gene, we evaluated the histopathological morphology of the liver and intestine, abundance and diversity of the intestinal flora, and function of the intestinal barrier in a mice model of liver cancer. Occludin, ZO-1, miRNA-144, and ZO-1 proteins were detected using real-time fluorescence quantitative PCR and a western blot. Conclusions Overexpression of miRNA-144 can inhibit the development of primary liver cancer, as well as improve the intestinal microecological environment and intestinal mucosal function in mice. The mechanism may be related to the up-regulation of mRNA and protein expression of Occludin and ZO-1.