Abstract Objectives To improve stability and shelf life, lyophilized formulations of 20(R)-Ginsenoside Rg3 liposomes (G-Rg3-Ls) were prepared. Methods Glucose, trehalose, sucrose, maltose, lactose, mannitol, inositol, hydroxypropyl-β-cyclodextrin and polyethylene glycol were used as single lyoprotectant and then compared in terms of their ability to protect lyophilized G-Rg3-Ls. Further, a glucose–mannitol complex was used to determine the optimal lyophilized preparation. The analysis of lyophilized liposomes or lyoprotectant was further investigated by scanning electron microscopy, thermogravimetry-differential thermal analysis, X-ray diffractometry and Fourier transform infrared spectroscopy. Cytotoxicity assay was used to assess the cyto-inhibition of freshly prepared and lyophilized liposomes. Key findings When the ratio of glucose–mannitol to phospholipids was 4 : 2 : 1 (w/w) the lyophilized G-Rg3-Ls exhibited good appearance, high DRR (86.52% ± 5.02%), small change in particle size (45.83 ± 0.50%) and short rehydration reconstruction time (8.3 ± 1.5 s). All indices were considerably better than those of each single protective agent. Results indicated that when the two lyoprotectants were combined, the stabilizing effect of glucose and shaping effect of mannitol were well maintained. The cyto-inhibition of freshly prepared and lyophilized G-Rg3 liposomes showed that lyophilization did not affect the bioactivity of G-Rg3. Conclusions The application of glucose–mannitol composite lyoprotectants can obtain a good G-Rg3 lyophilized preparation.