Although liposomes loaded with drug-cyclodextrin complexes have been proposed as drug delivery systems, the effects of cyclodextrins on the stability and fluidity of lipid membranes is still poorly understood. In this study, liposomes were prepared with the thin film hydration method and the effects of hydroxypropyl-β-cyclodextrin (HP-β-CD) on dipalmitoyl phosphatidyl choline (DPPC) membranes were investigated for the first time by differential scanning calorimetry, Raman spectroscopy and DPH fluorescence anisotropy at various HP-β-CD to DPPC molar ratios. HP-β-CD interacted with the choline head group of the phospholipids and with the DPPC alkyl chains, leading to an increase in the chain disorder. HP-β-CD loaded liposomes were also prepared with the ethanol injection method, using saturated and unsaturated phospholipids in the presence of cholesterol. HP-β-CD increased the disorder of unsaturated phospholipid bilayers, while no increase was observed for saturated ones. Furthermore, HP-β-CD present in the aqueous phase of saturated phospholipid liposomes protected them during freeze-drying.