Polyphyllin D (PD) is the active component from an Asian traditional medicinal herb Paris polyphylla, and has been found to hold significant anticancer activity in vivo or in vitro. However, the mechanism through which PD exerts its anticancer effects in triple-negative breast cancer (TNBC) remains unclear. Our study was presented to evaluate the anticancer effect and the potential mechanisms of PD in two TNBC cell lines, namely BT-549 and MBA-MB-231. Through comprehensively comparing the liquid chromatography-tandem mass spectrometry (LC-MS/MS) data of PD-treated and -untreated BT-549 and MBA-MB-231 cells, we found PD could activate oxidative phosphorylation pathway in BT-549 cells and inhibit spliceosome function in MDA-MB-231 cells, suggesting that the anticancer effect of PD may be cell type-specificity dependent. Moreover, we found that nodal modulator 2/3 (NOMO2/3) were down-regulated both in PD-treated BT-549 and MBA-MB-231 cells, suggesting NOMO2/3 may be the potential target of PD. Whether NOMO2/3 are the upstream modulators of oxidative phosphorylation pathway and Spliceosome need further validation. In conclusion, a comprehensive proteomics study was performed on PD-treated or untreated TNBC cells, revealing the anticancer mechanisms of PD.