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Gelatin is one of the most common natural macromolecules which is extensively used for preparing the nanoparticles. In the present study, type B gelatin-based nanoparticles were prepared using a twostep desolvation method and to optimize the nanoparticles, gelatin concentration, consumed acetone, temperature, and stirring speed were examined. Subsequently, vitamin D3 was encapsulated in obtained nanoparticles. Findings of the study indicated that the most optimum conditions for producing the nanoparticles with 20 mg/ml gelatin concentration were 15 ml acetone consumed volume, 40 °temperature and 1000 rpm stirring speed. The size of a produced particle under these conditions was reported as 88.6 nanometers. Also, the morphology of the obtained nanoparticle was spherical with a smooth surface which was observable through the scanning electron microscope. The size of the afore-mentioned particle increased to 177.2 nanometers followed by the addition of vitamin D3 to the produced nanoparticle which represents the encapsulation of the nanoparticle. [ABSTRACT FROM AUTHOR]