Sphingosine-1-phosphate (S1P) mediates several cytoprotective functions of HDL. ApoM acts as an S1P binding protein in HDL. Erythrocytes are the major source of S1P in plasma. After glomerular filtration apoM is endocytosed in the proximal renal tubules. Human or murine HDL elicited time- and dose-dependent S1P efflux from erythrocytes. Compared to HDL of wild type (wt) mice, S1P efflux was enhanced in the presence of HDL from apoM transgenic mice but not diminished in the presence of HDL from apoM knock-out (Apom-/-) mice. Artificially reconstituted and apoM-free HDL also effectively induced S1P efflux from erythrocytes. S1P and apoM were not measurable in the urine of wt mice. Apom-/- mice excreted significant amounts of S1P. ApoM was detected in the urine of mice with defective tubular endocytosis because of knock-out of LDL receptor related protein, chloride-proton exchanger ClC-5 (Clcn5-/-) or the cysteine transporter cystinosin. Urinary levels of S1P were significantly elevated in Clcn5-/- mice. In contrast to Apom-/- mice, these mice showed normal plasma concentrations for apoM and S1P. In conclusion, HDL facilitates S1P efflux from erythrocytes by both apoM-dependent and apoM-independent mechanisms. Moreover, apoM facilitates tubular reabsorption of S1P from the urine, however with no impact on S1P plasma concentrations.