Objective To construct a recombinant lentiviral vector harboring shRNA for mouse ribosomal protein S3a (RPS3a) and to analyze its effect on cell apoptosis. Methods Four pairs of shRNA sequences targeting mouse RPS3a mRNA were designed, and were ligated into pLLU2G-eGFP lentiviral vector. The recombinant plasmids were co-transfected with pLV/helper plasmids into 293T cells to package the recombinant lentivirus and the titers of the virus were determined. The lentivirus was introduced into RAW264.7 cells and levels of RPS3a mRNA and protein were detected by real-time PCR and Western blotting analysis, respectively. The apoptosis of RAW264.7 cells was detected by flow cytometry assays. Results PCR and DNA sequencing analysis confirmed that the recombinant lentivirus was successfully constructed and the virus titer was 6×10⁷-9×10⁷ TU/mL. Results of real-time PCR showed that the silencing efficiency of Lenti-shmRPS3a was 72.64%, and Western blotting analysis showed that RPS3a protein expression was decreased. Flow cytometry demonstrated that lentiviral-shRPS3a significantly increased cell apoptosis compared with the control group (P<0.05). Conclusion The constructed lentiviral vector harboring shRNA of RPS3a can efficiently silence RPS3a gene expression and promote cell apoptosis.