Specific knock-down of cellular gene expression using short, interfering RNAs (siRNAs) has become a powerful tool for functional genomics studies and a promising future therapeutic approach. However, recent studies have revealed that siRNAs can trigger an innate immune response upon intravenous administration in mice and transfection into purified immune cells by upregulating inflammatory cytokine levels. In this study, we demonstrate that transfection of siRNAs into several established human cancer cell lines can also induce inflammatory cytokine production regardless of the sequence of the siRNA used. The amount of inflammatory cytokine induction is cell type-specific, whereas the induction pattern is siRNA sequence-specific. We also show that, in a given cell type, different transfection reagents have different effects on inflammatory cytokine induction. Our results highlight the promiscuity of siRNA-triggered innate immune responses in human cancer cell lines and call for caution in the design and analysis of siRNA-based experiments.