Abstract: Objective To investigate the effect of the recombinant lentiviral vector for RNA interference(RNAi) of TLR4 gene on the expression of TLR4 gene and the growth of subcutaneous tumor of human hepatitis B virus-associated hepatocellular carcinoma in nude mice. Methods Four miR-TLR4 plasmid and a negative control plasmid were constructed, and a plasmid with obvious interference effect was selected for lentivirus packaging. BALB/C nude mice were inoculated subcutaneously with HepG2.2.15 cells to establish the subcutaneous tumor model of human hepatitis B virus-associated hepatocellular carcinoma. The mice were randomly divided into experimental group, negative control group and blank control group. TLR4 miRNA lentiviral particles, lentiviral particles carrying no effective sequence or NS(0.2 ml per time) were injected individually every two days. Tumor volume was measured to draw tumor growth curves. TLR4 expression was detected by semi-quantitative reverse transcription polymerase chain reaction (RTPCR), Western bolt and immunohistochemistry after 11 days. Results The mean rate of tumor growth in the experimental group was much slower than those in negative control group and blank control group(P<0.05); moreover, TLR4 mRNA and protein expression were significantly decreased in the experimental group(P<0.05). Conclusion Intra-tumor injection of TLR4 miRNA lentiviral vector has a significant inhibitory effect on the growth of subcutaneous tumor of human hepatitis B virus-associated hepatocellular carcinoma in nude mice.