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Immunogenic cell death (ICD) converts dying tumor cells into an endogenous vaccine, but clinically applicable pyroptosis-inducing agents remain scarce. Penicillide (PEN), a fungal metabolite, has not previously been examined for anticancer activity. PEN elicits potent, concentration-dependent cytotoxicity across six human cancer cell lines by triggering pyroptosis rather than apoptosis, necroptosis, ferroptosis, or autophagy. Electron microscopy observations showed that PEN causes noticeable damage to the cell membrane. PEN activates the canonical NLRP3/caspase-1/GSDMD axis, as evidenced by marked up-regulation of NLRP3, CASP1, and GSDMD transcripts, accumulation of cleaved GSDMD, and rescue of viability by their specific inhibitor and GSDMD siRNA. Mechanistically, PEN induces mitochondrial hyperpolarization followed by depolarization, depletes reduced glutathione, elevates malondialdehyde and reactive oxygen species, and activates NF-κB signaling via IκBα degradation and p65 nuclear translocation; all cytotoxic effects are reversed by the ROS scavenger N-acetylcysteine or the thiol-reducing agent dithiothreitol. PEN-treated cells exhibit hallmark features of immunogenic cell death-surface calreticulin exposure, nucleocytoplasmic HMGB1 translocation, and extracellular ATP release-and function as an effective prophylactic vaccine in Balb/c mice, delaying tumor outgrowth and augmenting intratumoral CD8⁺ cytotoxic T lymphocyte and CD11c⁺ cell infiltration. These findings establish PEN as a novel pyroptosis-inducing agent that couples oxidative stress and NF-κB signaling to elicit robust antitumor immunity, offering translational potential for ICD-based cancer immunotherapy.