Cephalostatin 1, a bis-steroidal marine natural product, has been reported to induce apoptosis without the requirement of an active caspase-8 or mitochondrial cytochrome c release and apoptosome formation. Here we show that despite the absence of these events, caspase-9 activation is essential for cephalostatin 1-induced apoptosis. Cephalostatin 1 initiates a rapid endoplasmic reticulum stress response characterized by phosphorylation of eukaryotic initiation factor-2 alpha-subunit and increased expression of the chaperone immunoglobulin heavy chain-binding protein GRP78 as well as the transcription factor C/EBP homologous protein (CHOP)/GADD153. Cephalostatin 1 activates apoptosis signal-regulating kinase 1 and c-Jun N-terminal kinase (JNK). However, this pathway does not play a major role in cephalostatin 1-induced apoptosis, as assessed by stable expression of a dominant negative apoptosis signal-regulating kinase 1. Importantly, the endoplasmic reticulum-associated caspase-4 is required and as shown by biochemical and genetic inhibition experiments, acts upstream of caspase-9 in cephalostatin-induced apoptosis.