Lactic acid bacteria, such as O. oeni, face stress that affects membrane fluidity. To overcome this, small HSP are produced and act as chaperones and lipochaperones. However, the molecular mechanisms involved in lipochaperone activity are still poorly understood, particularly with regard to the influence of membrane lipid composition on this activity. This work aimed at characterizing the influence of the lipid environment and the physiological state of the membrane on the structure and lipochaperone activity of Lo18. In this context, liposomes derived from O. oeni cultures in exponential, stationary and adapted growth phases at 8% alcohol, representing the physiological states of unstressed, stressed and stress-adapted bacterial membranes, were produced and characterized. These liposomes were then used to observe the effect of the physiological state of the membranes on the lipochaperone activity and structural changes of Lo18. The results allowed us to identify the preferential affinity of Lo18 for lipids composed of oleic acid and/or phosphatidylglycerol, the mainly phospholipid component of the membranes not yet adapted to stress. The impact of this membrane composition on the structure, lipochaperone activity and the insertion of Lo18 into the different types of liposomes showed that: (i) the presence of the membrane (regardless of its nature) induces a modification of the structure of Lo18; (ii) the insertion of Lo18 into the membrane and/or the lipochaperone activity of Lo18 is enhanced when the membranes are rich in oleic acid and/or phosphatidylglycerol. This work contributes to a better understanding of interaction mechanisms between sHSPs and bacterial membranes.