Background Lipid nanoemulsions that bind to low-density lipoprotein receptors can concentrate chemotherapeutic agents in tissues with low-density lipoprotein receptor overexpression and decrease the toxicity of the treatment. The aim of this study was to develop a new formulation using a lipophilic derivative of methotrexate, ie, didodecyl methotrexate (ddMTX), associated with a lipid nanoemulsion (ddMTX-LDE). Methods ddMTX was synthesized by an esterification reaction between methotrexate and dodecyl bromide. The lipid nanoemulsion was prepared by four hours of ultrasonication of a mixture of phosphatidylcholine, triolein, and cholesteryloleate. Association of ddMTX with the lipid nanoemulsion was performed by additional cosonication of ddMTX with the previously prepared lipid nanoemulsion. Formulation stability was evaluated, and cell uptake, cytotoxicity, and acute animal toxicity studies were performed. Results The yield of ddMTX incorporation was 98% and the particle size of LDE-ddMTX was 60 nm. After 48 hours of incubation with plasma, approximately 28% ddMTX was released from the lipid nanoemulsion. The formulation remained stable for at least 45 days at 4°C. Cytotoxicity of LDE-ddMTX against K562 and HL60 neoplastic cells was higher than for methotrexate (50% inhibitory concentration [IC 50 ] 1.6 versus 18.2 mM and 0.2 versus 26 mM, respectively), and cellular uptake of LDE-ddMTX was 90-fold higher than that of methotrexate in K562 cells and 75-fold in HL60 cells. Toxicity of LDE-ddMTX, administered at escalating doses, was higher than for methotrexate (LD 50 115 mg/kg versus 470 mg/kg; maximum tolerated dose 47 mg/kg versus 94 mg/kg) in mice. However, the hematological toxicity of LDE-ddMTX was lower than for methotrexate. Conclusion LDE-ddMTX was stable, and uptake of the formulation by neoplastic cells was remarkably greater than of methotrexate, which resulted in markedly greater cytotoxicity. LDE-ddMTX is thus a promising formulation to be tested in future animal models of cancer or rheumatic disease, wherein methotrexate is widely used.