The effect of lyophilization and rehydration medium on a liposome system for conjugated linoleic acid (CLA) delivery was studied. Liposomes were prepared by ethanol injection method employing soy phosphatidylcholine and CLA isomers 9c, 11t and 10t, 12c. Two rehydration media were assessed: a food simulant (distilled water) and the original medium used for liposome preparation (ethanol: water 1:9). Rehydrated liposomes were characterized in size, morphology, CLA encapsulation efficiency and membrane fluidity, at 3 and 30 days of cold storage. CLA loaded liposomes presented better stability and lower size than control liposomes without CLA through storage time, regardless of the rehydration medium. These facts could be related with spin label EPR measurements: CLA disordered the outer part of the lipid bilayer increasing fluidity, and ordered the inner part of the bilayer decreasing fluidity, which indicates a more packed membrane in the hydrophobic region. In all cases Transmission Electronic Microscopy images showed nanometric sized and oligo-lamellar vesicles. Liposomes preserved CIA isomers with high encapsulation efficiency: no differences were noticed between fresh and lyophilized samples. No influence of rehydration medium was noticed for the majority of the studied parameters. We have successfully developed efficient liposomal systems for bioactive compounds delivery in food applications.