The occurrence and the novel function of glyceraldehyde-3-phosphate dehydrogenase (GAPDH) in the extracellular space were studied. The extracellular GAPDH with the same molecular mass as the intracellular GAPDH was detected in the conditioned medium of mammalian cultured cell lines such as COS-7, HEK293, MCF-7, HepG2, PC-12, and Neuro-2a cells. Western blot analysis represented the occurrence of GAPDH, but not alpha-tubulin (an intracellular marker protein), in the conditioned medium of COS-7 cells. Furthermore, GAPDH was found in rat serum. These results indicate that GAPDH was secreted outside of the cells. Addition of GAPDH to the cultured medium of COS-7, HEK293, and HepG2 cells allowed cells to undergo morphological changes. In COS-7 cells, the extracellular GAPDH inhibited cell spreading without influencing the cell growth. Western blot and immunofluorescent microscopy analyses revealed that the extracellular GAPDH bound to COS-7 cells in time- and dose-dependent manners. However, a mutant substituting Ser for Cys at position 151 of GAPDH resulted in no binding to the cells, no decreased cell-spreading efficiency and no cell morphological changes. These results indicate that the Cys151 was involved in the binding of GAPDH to cells and the GAPDH-inhibited cell spreading.