Cancer nanotherapeutics are progressing rapidly with innovative drug delivery systems to replace conventional delivery systems. Although, antitumor activity of zerumbone (ZER) has been reported, but low water solubility (1.296 mg/L at 25ºC) of ZER plagued its oral bioavailability, limited its transportation during oral administration and impaired its delivery to targeted organs and tissues. ZER-loaded nanostructured lipid carrier (NLC) was successfully prepared and developed by high pressure homogenization (HPH) technique. Physicochemical characterization including particle size, polydipersity index, zeta potential, pH, entrapment efficiency, loading capacity, and in vitro drug release using Zetasizer, reverse phase high performance liquid chromatography (RP- HPLC), transmission electron microscopy (TEM), wide angle x-ray diffraction (WAXR), differential scanning colarimeter (DSC) and Franz Diffusion Cell (FDC) system analysis has been performed. Moreover, the anticancer efficacy of ZER-NLC on human acute lymphocytic leukemia (ALL) was also studied in vitro. However, there has been no available information of ZER-NLC affects murine leukemia cells in vitro. In this study, in vitro effects of ZER-NLC on murine leukemia WEHI-3B cells were investigated. The results demonstrated that the growth of leukemia cells in vitro was inhibited by ZER-NLC using MTT, Hoechst 33342, Annexin V and cell cycle. In conclusion, we suggest that ZER-NLC might be helpful for improving the usefulness of anticancer agents in the therapy of leukemia.