Shiga toxin (Stx) has an A1-B5 subunit structure, and the A subunit is an RNA N-glycosidase that inhibits cellular protein synthesis. We previously reported that in Caco-2 cells Stx induced cytokines and that the RNA N-glycosidase activity was essential for the cytokine induction. It is known that the binding of the Stx-B subunit to its receptor glycolipid, Gb3, mediates an A subunit-independent signal in some types of cells, but the involvement of this signal in the cytokine induction is unclear. In this study, we investigated whether RNA N-glycosidase itself induces cytokines. IL-8 production was enhanced by Stx, ricin, and modeccin, three toxins that inhibit protein synthesis through an identical RNA N-glycosidase activity, but not by two other types of protein synthesis inhibitors, diphtheria toxin and cycloheximide. The RNA N-glycosidase-type toxins showed a similar induction pattern of cytokine mRNAs. Brefeldin A, a Golgi apparatus inhibitor, completely suppressed the cytokine induction by the toxins. Analysis by using inhibitors of toxin binding and also Stx-B subunit showed that the cytokine-inducing activity was independent of Gb3-mediated signaling. These results indicate that RNA N-glycosidase itself induces the cytokine production and that intracellular transport of toxins through the Golgi apparatus is essential for the activity.