Strategy, Management and Health Policy Enabling Technology, Genomics, Proteomics Preclinical Research Preclinical Development Toxicology, Formulation Drug Delivery, Pharmacokinetics Clinical Development Phases I-III Regulatory, Quality, Manufacturing Postmarketing Phase IV The aim of the study was to develop a novel oil/water microemulsion system to increase the cytotoxic effect of mitomycin C (MMC) on human lung cancer cell lines through comparison to the conventional MMC solution. The microemulsion formulation was composed of soybean oil, lecithin, Tween 80, ethanol, and water. Characterization of the microemulsions was carried out by means of particle size, viscosity, conductivity, storage stability, in vitro drug release, and in vitro hemolysis. Putative anticancer activity was determined using Calu1 and A549 carcinoma cell lines with an XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] cell proliferation assay. Drug release from the MMC-loaded microemulsion was sustained for more than 5?h while release from MMC solution was completed within 2?h. Based on the results of cytotoxicity study, a higher anticancer effect was observed with mitomycin C-loaded microemulsion (MMC-M), with IC 50 values being approximately twofold to fourfold higher than that seen with the MMC solution on Calu1 and A549 carcinoma cell lines, respectively. In conclusion, MMC microemulsion has several advantages including slower drug release, a more pronounced anticancer effect at lower MMC doses, potentially leading to lower systemic toxicity potential if leakage occurs from the tumor site.