The development of efficient liposomal encapsulations of proteins for pharmaceutical applications is limited by several factors: their high molecular weight, interactions with surrounding substances, or the generally lower stability compared to small molecules. In this work, various liposomal formulations were prepared using the thin-film hydration method followed by extrusion, to investigate their suitability for the encapsulation of the plant-derived antitumoral mistletoe lectin-1 (ML-1). This can be significantly optimized by exploiting its preferential binding to galactose-containing structures, such as modified lipids integrated into the liposomal bilayer. Incorporation of the galactosylated lipid DSPE-PEG2k-Gal into the membrane significantly enhanced the overall recovery rate and encapsulation efficiency of ML-1, attributed to its affinity for the functionalized component. Compared to non-functionalized liposomes, a 2-fold to 4-fold increase in percentage encapsulation efficiency was observed. The galactosylated lipid optimized the ratio of encapsulated to surface-adsorbed ML-1 and facilitated its preferential localization within the core of the liposomes. A strong correlation was identified between the number of entrapped ML-1 molecules per liposome and the degree of galactosylation. The formulations demonstrated high in vitro cytotoxicity, as exemplified with murine colon-26 carcinoma cells, with the galactose-functionalized liposomes achieving an IC50 value comparable to free ML-1. This strategy presents significant potential for developing more efficient and targeted liposomal formulations of pharmaceutical proteins with specific affinities to tailored lipid components, advancing drug delivery technologies, and improving therapeutic options for cancer treatment.