RNA interference (RNAi) is one of the most promising tools for deciphering the human genome and has great therapeutic potential. However, its high target specificity limits its efficiency for therapeutic protection from viruses with high rates of genetic mutation. This limitation may be overcome by the expression of long hairpin RNAs (lhRNAs). Indeed, lhRNAs have been shown recently to have increased efficacy over short interfering RNAs (siRNAs) as protective antiviral agents. Here, we investigate the expression of lhRNAs and demonstrate unintended effects. We show that overexpressed lhRNAs are exported to the cytoplasm. As a consequence, we detect activation of innate immune signaling pathways by lhRNAs. With growing concerns about the complexity of cytoplasmic detection of dsRNAs by the innate immune machinery, this work highlights the need for closer scrutiny when using lhRNAs as potential antiviral agents.