The aim of this study was to develop and characterize methotrexate-loaded polymeric nanoparticles by nanoprecipitation technique. Eudragit? S 100 and ethyl cellulose were employed to develop methotrexate-loaded nanoparticles by nanoprecipitation technique. Six different formulations (f1, f2, f3, f4, f5 and f6) were prepared with each polymer by varying the drug to polymer ratios (1:1, 1:1.5, 1:2, 1:3, 1.5:1 and 2:1). Dimethyl sulfoxide (DMSO) was used as a solvent and Tween? 20 as a surfactant. Among the six formulations of polymeric nanoparticles prepared by nanoprecipitation technique, F4 formulation was considered as a best formulation with each polymer. Based on comparison results of mean particle diameter, zeta potential, drug content and entrapment efficiency, Eudragit? S 100 was considered to be the most suitable polymer for preparation of methotrexate-loaded nanoparticles by nanoprecipitation technique. Based upon the evaluation studies, the best formulation was characterized for scanning electron microscopy (SEM), particle size, zeta potential and anti-cancer activity in MCF-7 cell line by MTT assay.