Biodegradable polymeric nanoparticles have gained importance since they improve the therapeutic value of various water insoluble drugs and bioactive molecules by improving bioavailability, solubility and retention time. Nanosized drug delivery systems provide enhanced pharmacokinetic and pharmacodynamic control compared to free drugs. In this study, cationic gelatin nanoparticles were first prepared by the two step desolvation method and then their in vitro Paclitaxel (Ptx) delivery ability was evaluated. Resulting nanoparticles were analyzed for their in vitro cytotoxic effect in two different cell lines. The experimental results showed that spherical gelatin nanoparticles had a zeta potential of approximately +15.5 mV and the average size of the particles was 10.3±0.42 nm. Ptx release from gelatin nanoparticles exhibited an initial burst release followed by diffusion of the drug in a sustained manner and finally polymer erosion occured up to 38 days. In vitro cell viabilitiy analysis showed that Ptx loaded gelatin nanoparticles inhibited the growth of A549 and HT29 cells more effectively than the free drug. Based on these findings, it can be stated that cationic gelatin nanoparticles may be a promising therapeutic strategy for the treatment of many cancers.