We have previously shown that transforming growth factor-β1 (TGF-β1)-induced apoptosis in FaO hepatoma cells is mediated by cytochrome c release, apoptosome formation, and caspase activation. Although TGF-β1 acts via the SMAD signaling pathway to initiate de novo gene transcription, little is known about the downstream gene targets that are involved in the regulation of apoptosis. Therefore, in this study, we used in-house microarrays (∼5500 genes) to identify pathway-specific gene clustering in TGF-β1-treated cells. A total of 142 genes showed time-dependent changes in expression during TGF-β1-induced apoptosis. The polycaspase inhibitor benzyloxycarbonyl-VAD-fluoromethyl ketone, which, on its own, had no effect on gene transcription, blocked TGF-β1-induced cell death and significantly altered the expression of 261 genes, including 185 down-regulated genes. Cluster analysis identified up-regulation of early response genes (0-4 h) encoding for the extracellular matrix and cytoskeleton, including the pro-apoptotic CTGF gene, and delayed response genes (8-16 h), including pro-apoptotic genes. A second delayed response cluster (44 genes) was also observed when TGF-β1-induced caspase activation was blocked by benzyloxycarbonyl-VAD-fluoromethyl ketone. This cluster included genes encoding stress-related proteins (e.g. Jun, ATF3, TAB1, and TANK), suggesting that their up-regulation may be in response to secondary necrosis. Finally, we identified an early response set of nine down-regulated genes that are involved in antioxidant defense. We propose that the regulation of these genes by TGF-β1 could provide a molecular mechanism for the observed elevation in reactive oxygen species after TGF-β1 treatment and may represent the primary mechanism through which TGF-β1 initiates apoptosis.