​Gemcitabine (dFdC) is a widely used prodrug against pancreatic cancer and has been investigated against pancreatic, glioblastoma and breast cancer. However, the nature of gemcitabine hinders its incorporation into nanocarriers at high loading levels. This work focuses for the first time on optimizing gemcitabine-loaded stearic acid-based niosomes for enhanced anti-tumor efficacy. The antitumor effect of the optimized formula (dFdC −9 mM ⊂ Nio) was studied against three undruggable cancer cell lines: pancreatic cancer (PANC-1), glioblastoma (U-87), and triple negative breast cancer (MDA-MB-231), and against normal fibroblasts (HDF). The size of dFdC −9 mM ⊂ Nio formula was 151.3 ± 3.3 nm, the encapsulation efficiency was 68.83 ± 0.08%, and the drug loading capacity was 16.5 ± 0.7%. Cellular uptake of dFdC −9 mM ⊂ Nio by PANC-1 was found to increase 3-fold relative to the untreated cancer cell​