Polyphyllin D (PD) is a potent cytotoxic saponin found in Paris polyphylla. In the present study, bioinformatic, proteomic and transcriptomic analyses were performed to study the mechanisms of action of PD on human nonsmall cell lung cancer (NSCLC) cell line (NCI-H460). Using a gene expression-based bioinformatic tool (connectivity map), PD was identified as a potential ER stress inducer. Our proteomic and transcriptomic analyses revealed that PD treatment led to upregulation of typical ER stress-related proteins/genes including glucose-regulated protein 78 (BiP/GRP78) and protein disulfide isomerase (PDI). In particular, elevated expression of C/EBP homologous transcription factor (chop) and activation of caspase-4 occurred at early time point (8 h) of PD treatment, signifying an initial ER stress-mediated apoptosis. Induction of tumor suppressor p53, disruption of mitochondrial membrane, activation of caspase-9 and caspase-3 were detected upon prolonged PD treatment. Collectively, these data revealed that PD induced the cytotoxic effect through a mechanism initiated by ER stress followed by mitochondrial apoptotic pathway. The ability of activating two major pathways of apoptosis makes PD an attractive drug lead for anticancer therapeutics.