Liposomes were proposed as drug vector systems in the treatment of many diseases. The following characteristics recommend the liposomes as attractive candidates for drug transportation: solubilisation, duration of action, targeting potential and internalisation. Methotrexate, a folate antagonist, was originally developed as an antineoplastic agent and subsequently used in inflammatory and/or immunosuppressive diseases. Its side effects have led researchers to direct their efforts to reduce toxicity, while maintaining efficacy of methotrexate. Liposomes with methotrexate as such, as well as its disodium salt, were prepared using two methods. The liposomes were characterized in terms of structure, size, degree of poly‐dispersion and encapsulation efficiency. The effect of methotrexate incorporated in liposomes has been investigated in vitro on human lymphoblastic cell line K562. Methotrexate incorporated into liposomes moderately reduces the proliferation of K562 cells, but significantly inhibits RNA synthesis. The cellular activation is probably the main target of the drug and not the neoplastic proliferation of cells. The methotrexate liposomes exhibited significant anti‐inflammatory activity and showed reduced toxicity. Given that the encapsulating of the drug in vector systems may result in the increasing concentration at the site of action, the methotrexate liposomes represent a targeted therapy with an optimized therapeutic efficacy—risk toxicity ratio.