We previously showed that polyphyllin D (PD) produced a stronger apoptotic effect in R-HepG2 with multi-drug resistance (MDR) than that in its parent HepG2 cells without MDR. In this study, PD was found to elicit mitochondrial fragmentation in live cells by using total internal reflection fluorescence microscopy (TIRFM). When mitochondria were isolated and treated directly with PD, a stronger swelling, deeper transmembrane depolarization, and more apoptosis-inducing factor (AIF) release were observed from the mitochondria of R-HepG2 than that of HepG2. These observations suggest that PD is a potent anti-cancer agent that bypasses MDR and elicits apoptosis via mitochondrial injury.