A succinoyl trehalose lipid produced by Rhodococcus sp. behaves as a biological surfactant and also displays various interesting biological activities. Trehalose lipid has been shown to have a great tendency to partition into phospholipid membranes; therefore, the characterization of its interaction with biological membranes is of central importance. In this work, human red blood cells have been used as an experimental model. Trehalose lipid causes the swelling of human erythrocytes followed by hemolysis at concentrations well below its critical micellar concentration. Kinetic measurements show that, upon addition of trehalose lipid, K release precedes that of hemoglobin. Osmotic protectants of the appropriate size added to the external medium make it possible to avoid hemolysis. The results indicate that trehalose lipid causes the hemolysis of human erythrocytes by a colloid-osmotic mechanism, most likely by formation of en ha need permeability domains, or "pores" enriched in the biosurfactant, within the erythrocyte membrane. Scanning electron microscopy shows trehalose lipid-induced spherocytosis and echinocytosis of red blood cells, which tits well within the framework of the bilayer-couple hypothesis. The presented results contribute to establishing a molecular basis for the biological properties of this trehalose lipid biosurfactant.