The  Schizosaccharomyces pombe shu1  +  gene encodes a cell-surface protein required for assimilation of exogenous heme. In this study, shaving experiments showed that Shu1 is released from membrane preparations when spheroplast lysates are incubated with phosphoinositide-specific phospholipase C (PI-PLC). Shu1 cleavability by PI-PLC and its predicted hydropathy profile strongly suggested that Shu1 is a glycosylphosphatidylinositol-anchored protein. When heme biosynthesis is selectively blocked in  hem1 Δ mutant cells, the heme analog zinc mesoporphyrin IX (ZnMP) first accumulates into vacuoles and then subsequently, within the cytoplasm in a rapid and Shu1-dependent manner. An HA 4 -tagged  shu1  +  allele that retained wild-type function localizes to the cell surface in response to low hemin concentrations, but under high hemin concentrations, Shu1-HA 4  re-localizes to the vacuolar membrane. Inactivation of  abc3  + , encoding a vacuolar membrane transporter, results in  hem1 Δ  abc3 Δ mutant cells being unable to grow in the presence of hemin as the sole iron source. In  hem1 Δ  abc3 Δ cells, ZnMP accumulates primarily in vacuoles and does not sequentially accumulate in the cytosol. Consistent with a role for Abc3 as vacuolar hemin exporter, results with hemin-agarose pulldown assays showed that Abc3 binds to hemin. In contrast, an Abc3 mutant in which an inverted Cys-Pro motif had been replaced with Ala residues fails to bind hemin with high affinity. Taken together, these results show that Shu1 undergoes rapid hemin-induced internalization from the cell surface to the vacuolar membrane and that the transporter Abc3 participates in the mobilization of stored heme from the vacuole to the cytosol.