RNA interference (RNAi) has recently emerged as a promising antiviral technique in vertebrates. Although most studies have used exogenous short interfering RNAs (siRNAs) to inhibit viral replication, vectors expressing short hairpin RNAs (shRNA-mirs) in the context of a modified endogenous micro-RNA (miRNA) are more efficient and are practical for in vivo delivery. In this study, replication competent retroviral vectors were designed to deliver shRNA-mirs targeting subgroup B avian leukosis virus (ALV), the most effective of which reduced expression of protein targets by as much as 90% in cultured avian cells. Cells expressing shRNA-mirs targeting the tvb receptor sequence or the viral env(B) sequence significantly inhibited ALV(B) replication. This study demonstrates efficient antiviral RNAi in avian cells using shRNA-mirs expressed from pol II promoters, including an inducible promoter, allowing for the regulation of the antiviral effect by doxycycline.