The aim of this study was to design and fabricate nanostructured lipid carrier (NLC) for the potential oral delivery of decitabine (DCB). NLC was prepared by cold homogenization technique and optimized by the Box-Behnken experimental design. It was further characterized by particle size, zeta potential, transmission electron microscopy (TEM), atomic force microscopy (AFM), differential scanning calorimetry (DSC), X-ray diffraction (XRD), in vitro release study, and stability study. Moreover, ex vivo and in vivo efficacy of the NLC was assessed by gut permeation study, γ scintigraphy imaging, and MTT assay. NLC was found to have particle size (116.64 ± 6.67 nm), zeta potential (-31.8 ± 0.96 mV) and sustained drug release (80.23 ± 4.67%) up to 24h. TEM and AFM proved that the particles were spherical in shape and smooth surface. DSC and XRD studies had demonstrated the reduced crystallinity and stability enhancing effect of the NLC. Stability studies revealed the changes in the observed parameters up to 45 days were not significantly differences (p>0.05). Ex vivo gut permeation study showed 4-folds increment in the permeation of drug compared with the plain drug solution. γ Scintigraphy imaging and MTT assay results inferred that DCB loaded NLC possesses excellent cytotoxic activity against cancer cells. Thus, NLC holds high potential for the oral delivery of DCB to treat cancer cells and future prospects for the industrial purpose.