This study investigated the cytotoxic effects of gemcitabine-loaded solid lipid nanoparticle (Gem-SLN) on the patient-derived primary pancreatic cancer cell lines (PPCL-46) and MiaPaCa-2. Different SLN formulations were prepared from glyceryl monostearate (GMS), polysorbate 80 (Tween® 80) and poloxamer 188 (Pol 188) as surfactants using a cold homogenization method. Gem-SLN was characterized for particle size and charge distribution, entrapment efficiency and loading capacity. Fourier Transform Infra-Red (FTIR) spectroscopy was used to verify Gem and SLN interaction while differential scanning calorimetry (DSC) was used to acquire thermodynamic information on Gem-SLN. Cytotoxicity studies was conducted on PPCL-46 cells and Mia-PaCa-2 cells. Among the different Gem-SLN formulations prepared, Gem-SLN was selected based on entrapment efficiency (EE) of Gem, loading efficiency of Gem, cytotoxicity and rate of Gem release. Growth inhibition of Gem-SLN-treated PPCL-46 culture (IC (2D) =27± 5 μM; IC (3D) = 66 ± 2 μM) was remarkably higher than gemcitabine hydrochloride (GemHCl)-treated PPCL-46 culture (IC (2D) =126±3 μM; IC (3D) =241±3 μM). Similar trend of higher Gem-SLN inhibition in MiaPaCa-2 culture was found (IC (2D) =56±16 μM; IC (3D) =127±4 μM) compared with GemHCl-treated Mia-PaCa-2 culture (IC (2D) =188±46 μM; IC (3D) =254±52 μM). The anticancer activity of Gem-SLN was significantly more effective than GemHCl in PPCL-46 compared to Mia-PaCa-2 cancer cells. Schematic diagram for preparation of Gem-SLN through cold homogenization and methods for characterization and studies.