Objectives: Cationic lipid complexes bind to angiogenic endothelial cells of solid tumours and microvessels of chronic inflammatory tissue. Methotrexate (MTX) is one of the drugs used in the therapy of rheumatoid arthritis (RA); it is applied systemically but can have serious side-effects. The aim of this study was to investigate the impact of MTX encapsulated in cationic liposomes (EndoMTX) in comparison to treatment with free MTX.Method: We used an antigen-induced arthritis (AiA) model and investigated the leucocyte– and platelet–endothelial cell interaction in arthritic female C57/Bl6 mice and in healthy controls. The arthritic animals were divided into four different groups receiving either trehalose, free MTX, EndoMTX placebo, or EndoMTX. These parameters and functional capillary density (FCD) were measured and assessed by intravital microscopy (IVM). We controlled clinical parameters such as the knee joint diameter (KJD) throughout the observation period.Results: Animals treated with EndoMTX showed a significant and superior reduction in leucocyte– and platelet–endothelial cell interaction, FCD, and KJD. Free MTX or empty liposomes also showed a reduction in these parameters but not to a significant level. FCD decreased in the EndoMTX group in comparison to using free drugs or empty carrier-like liposomes.Conclusions: This study demonstrates the advantage of using MTX encapsulated in cationic liposomes in contrast to free and generic MTX, with a higher efficacy in anti-inflammatory and anti-angiogenic abilities. Targeting with cationic liposomes may be a promising treatment option and should be elucidated in further experiments regarding dose reduction and side-effects due to MTX usage.