Objective: To construct and recombine a lentivirus-mediated short hairpin RNA (shRNA) vector targeting NUP88 gene and to observe the effect of silencing NUP88 gene on the biological behavior of MCF-7 cells by RNA interference (RNAi) technology, so as to find a new target for the treatment of breast cancer. Materials and Methods: The recombinant lentiviral vector of NUP88-shRNA was constructed and then transfected into MCF-7 cells. Then, the changes of biological behavior of MCF-7 cells were detected by reverse transcription polymerase chain reaction (RT-PCR), Western-blot, methyl thiazolyl tetrazolium (MTT), flow cytometry, and Transwell assay. Results: The results of RT-PCR and Western-blot showed that the expression of NUP88 mRNA and protein was significantly reduced in NUP88-shRNA group, as compared with that in the blank and negative groups (p < 0.01). The proliferation of MCF-7 cells in the NUP88-shRNA group was prominently reduced after transfected with lentivirus-NUP88-shRNA (p < 0.05). Flow cytometry results showed that the apoptosis rate increased significantly in the NUP88-shRNA group as compared with the other two groups (p < 0.05). After routine culture for 24 hours, the transmembrane cells in the NUP88-shRNA group were notably less than that in the blank or negative control group (p < 0.05). Conclusion: Recombinant NUP88-shRNA lentivirus can successfully inhibit the expression of NUP88 gene in MCF-7 cells and consequently inhibit the proliferation and invasive ability of human breast cancer cells.