Aims: The purpose of the present study was to evaluate the ability of calcic and magnesic-sulphate-sulphurous spring mineral water (SMW) to reduce total and erythrocyte plasma membrane cholesterol in rats fed a cholesterol-rich diet. Methods: By using different fluorescent probes, 1,6-diphenyl-1,3,5-hexatriene (DPH), 1-[4-(trimethylamino)phenyl]-6-phenylhexa-1,3,5-triene (TMA-DPH) and Laurdan, a possible correlation was evaluated between cholesterol content changes of the membranes and membrane fluidity. Results: The results clearly show that hydropinic treatment reduced cholesterol level. No significant change was observed in DPH and TMA-DPH anisotropy values. By using Laurdan, an increase was observed in generalized polarization (GP340) in both groups of rats (the one that drank tap water and the one that drank SMW) compared with the controls. The rate of haemolysis was reduced in those erythrocyte suspensions in which cholesterol was increased, even if the enzymatic activity of glutathione peroxidase and catalase decreased. Conclusion: Hydropinic treatment with SMW is of interest from the biomedical point of view, because it could represent an alternative approach in interventions aimed at ameliorating biological injury deriving from hypercholesterolaemia.