The objective of this study was to prepare the microemulsion of methotrexate ( M- MTX) for oral use and to investigate the suppressive effect of MTX-loaded microemulsion on MCF-7 human breast cancer cells. At the same time this effect of M- MTX was compared with those of a solution of the drug ( Sol-MTX). Microemulsion was composed of soybean oil as oil phase, a mixture of Cremophore EL and Span 80 as surfactants, and isopropyl alcohol as co-surfactant, and 0.2 N NaOH as the aqueous phase. MTX was added into microemulsion at the last stage. We clearly demonstrated that M- MTX had a significant cytotoxic effect on breast cancer cell lines and the cytotoxic effect of M-MTX was significantly more than that of solutions ( p < 0.05) and IC50 value for M- MTX was 40 ng/mL. We also examined M- MTX and Sol- MTX on a model biological environmental model. For this purpose a gastrointestinal cell culture model, the Caco-2 cell line, was used to investigate the cytotoxic effects of the polymeric carrier and its effect on the cell monolayer integrity. The differences between the viability of cells for M- MTX and Sol- MTX were significantly different when applied to ANOVA according to 2 x 8 factorial randomized design ( p: 0.016; for a: 0.05, power : 0.695). According to the in vitro cytotoxicity studies, we concluded that when MTX was incorporated into the microemulsion (M- MTX), which is a new drug carrier system, it suppresses tumour cell growth on multiple tumor lines. These results indicate that M- MTX may exert a low cytotoxic effect on normal cells and may be effective as an antitumor agent that induces apoptosis.