Phospholipase B (Plb1) is secreted by pathogenic fungi and is a proven virulence determinant in Cryptococcus neoformans. Cell-associated Plb1 is presumptively involved in fungal membrane biogenesis and remodelling. We have also identified it in cryptococcal cell walls. Motif scanning programs predict that Plb1 is attached to cryptococcal membranes via a glycosylphosphatidylinositol (GPI) anchor, which could regulate Plb1 export and secretion. A functional GPI anchor was identified in cell-associated Plb1 by (G)PI-specific phospholipase C (PLC)-induced release of Plb1 from strain H99 membrane rafts, and inhibition of GPI anchor synthesis by YW3548, which prevented Plb1 secretion and transport to membranes and cell walls. Plb1 containing -1,6-linked glucan was released from H99 (wildtype strain) cell walls by -1,3 glucanase, consistent with covalent attachment of Plb1 via -1,6-linked glucans to -1,3-linked glucan in the central scaffold of the wall. Naturally-secreted Plb1 also contained -1,6-linked glucan, confirming that it originated from the cell wall. Plb1 maintains cell wall integrity since a H99 deletion mutant, PLB1, exhibited a morphological defect and was more susceptible than H99 to cell wall disruption by SDS and Congo red. Growth of PLB1 was unaffected by caffeine, excluding an effect of Plb1 on cell wall biogenesis-related signalling pathways. Environmental (heat) stress caused Plb1 accumulation in cell walls, with loss from membranes and reduced secretion, further supporting the importance of Plb1 in cell wall integrity. This is the first demonstration that Plb1 contributes to fungal survival by maintaining cell wall integrity and that the cell wall is a source of secreted enzyme.