In addition to its well described function as an enzymatic inhibitor of specific caspases, X-linked inhibitor of apoptosis (X-linked IAP or XIAP) can function as a cofactor in Smad, NF-kappaB, and JNK signaling pathways. However, caspases themselves have been shown to regulate the activity of a number of signaling cascades, raising the possibility that the effect of XIAP in these pathways is indirect. Here we examine this question by introducing point mutations in XIAP predicted to disrupt the ability of the molecule to bind to and inhibit caspases. We show that whereas these mutant variants of XIAP lost caspase-inhibitory activity, they maintained their ability to activate Smad, NF-kappaB, and JNK signaling pathways. Indeed, the signaling properties of the molecule were mapped to domains not directly involved in caspase binding and inhibition. The activation of NF-kappaB by XIAP was dependent on the E3 ubiquitin ligase activity of the RING domain. On the other hand, the ability of XIAP to activate Smad-dependent signaling was mapped to the third baculoviral IAP repeat (BIR) and loop regions of the molecule. Thus, the anti-apoptotic and signaling properties of XIAP can be uncoupled.