In this study, magnetic nanoparticles (MNPs) coated with glycine (F-Gly NPs) and conjugated with methotrexate (MTX) (F-Gly-MTX NPs) were synthesized through a coprecipitation method followed by amidation reaction between the carboxylic acid end groups on MTX and the amine groups on the MNPs surface and studied its cytotoxic effect in vitro. The successful conjugating of MTX onto the nanoparticles (NPs) was confirmed by X-ray diffraction, thermogravimetric analysis, differential scanning calorimetry, Fourier transform infrared spectroscopy, vibrating sample magnetometer, and transmission electron microscopy techniques. The results showed that the average size was 46.82 ± 5.03 nm. This target drug delivery system is dependent on the release of the MTX within the lysosomal compartment. Hemolysis assay and cytotoxicity study results on HFF-2 and HEK-293 cell lines show that as prepared MNPs are biocompatible. The cytotoxicity of void of the MTX and F-Gly-MTX NPs were compared to each other by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay of the treated MCF-7 cell line. Enzymatic release studies exhibited the release of the MTX via peptide bond cleavage in the presence of proteinase K. These studies specify that the F-Gly-MTX NPs have a very remarkable anticancer effect, for breast cancer cell line. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 1646-1654, 2018.