RNA interference (RNAi) has exhibited huge potentials on anti-HIV-1 therapy research. The obtainment of RNAi element targeting to HIV-1 highly effectively and specifically was crucial for relevant research. Recent reports had described that microRNAs (miRNAs) posses more characteristics of inhibition and expression mechanisms than small interfering RNAs (siRNAs). In this study we explored the construction of artificial miRNA targeting to HIV-1 effectively and specifically. Sixteen siRNAs sequences were selected based on the conserved regions in the HIV-1 pol gene. ShRNA expression vectors were co-transfected with HIV-1 clone pNL4-3 to evaluate the abilities of siRNAs to inhibit HIV-1 expression. The pol1026 sequence was selected from candidates. The target sequence in the stem-loop structure of the well-characterized native miR-30a was replaced with pol1026 sequences, and the artificial miRNA expression vectors were co-transfected with the HIV-1 clone pNL4-3, results showed that HIV-1 can be effectively inhibited by miR-1026E. Target specificity of miR-1026E was confirmed by co-transfection assay with reporter plasmids containing different target sequences. The miR-1026E expression element was then inserted into Lentivirus which was used as a vector to transduce the MT-4 cells, MT-4-miR1026E expressing miR-1026E stably was cloned from transduced cells. The MT-4-miR1026E cell effectively inhibited HIV-1 replication in vitro. And the intracellular miR-181 and miR-16 expression levels and stat1 mRNA levels were not affected by the expression of miR-1026E in MT-4-miR1026E cells. miR-1026E is a promising candidate for future research.