RNA interference (RNAi) is a sequence-specific RNA degradation process. To inhibit feline immunodeficiency virus (FIV) replication by RNAi, we generated a lentiviral vector expressing a short hairpin RNA (shRNA) that targeted the gag gene of FIV (shGag). shGag transfer significantly inhibited viral replication in cell lines that were chronically infected with FIV, i.e., the 3201/UK8 low, 3201/UK8 high, FL4, and CRFK/FIV cell lines. Moreover, 3201 cells were transduced with the lentiviral vectors and then inoculated with FIV. Although the amount of FIV proviral DNA in shGag-transduced cells was similar to that in the cells transduced with unrelated shRNA or mock-transduced cells, the amount of reverse transcriptase (RT) activity was significantly reduced in the culture supernatant of shGag-expressing cells from 15 to 27 days after inoculation. Thirty days after inoculation, no significant difference was observed in the RT activities but virus with a mutation in the target region of shGag was detected in approximately 21% of the replicated viruses. Therefore, abolishment of the silencing effect of shGag may be due to reasons other than the emergence of escape mutants. These results are useful for developing an RNAi-based gene therapy strategy for controlling FIV infection.