Long (27-29-bp dsRNA) Dicer-dependent substrates have been identified as potent mediators of RNAi-induced gene knockdown in HEK293 and HeLa cells. As the lengths of these molecules are reported to be below the threshold generally regarded as necessary for induction of the mammalian interferon (IFN) response, these long siRNA are being considered as RNAi substrates in both research and therapeutic settings. In this report, we demonstrate that >23-bp dsRNA can influence cell viability and induce a potent IFN response (highlighted by a strong up-regulation of the dsRNA receptor, Toll-like receptor 3) in a cell type-specific manner. This finding suggests that the length threshold for siRNA induction of the IFN response is not fixed but instead varies significantly among different cell types. Given the diversity of cell types that comprise whole organisms, these findings suggest great care should be taken when considering length variations of dsRNA molecules for RNAi experimentation, especially in therapeutic applications.