Our previous report has shown that honokiol (HNK), a constituent of Magnolia officinalis, induces a novel form of non-apoptotic programmed cell death in human leukemia NB4 and K562 cells. In this study, we further explored the relationship between the cell death pathway and cytoplasmic vacuolization and studied the underlying mechanism of leukemia cell death mediated by honokiol. The results showed that low concentrations of honokiol activated an novel alternative cell death fitted the criteria of paraptosis, such as cytoplasmic vacuolization derived from endoplasmic reticulum swelling, lack of caspase activation, and lack of apoptotic morphology. Results further indicated that the cell death was time- and concentration-dependent. In addition, honokiol-induced paraptosis did not involve membrane blebbing, chromatin condensation and phosphatidylserine exposure at the outer of the plasma membrane. The mechanism of the cell death may be associated, at least in part, with the increased generation of reactive oxygen species. Further analysis showed that honokiol induces cell death predominantly via paraptosis and to a certain extent via apoptosis in NB4 cells, and predominantly via apoptosis and to a certain extent via paraptosis in K562 cells. These observations suggest that cell death occurs via more than one pathway in leukemia cells and targeting paraptosis may be an alternative and promising avenue for honokiol in leukemia therapy.