Previous studies have shown that yeast glycosylphosphatidylinositol-anchored proteins (GPI-APs) and other secretory proteins are preferentially incorporated into distinct coat protein II (COPII) vesicle populations for their transport from the endoplasmic reticulum (ER) to the Golgi apparatus, and that incorporation of yeast GPI-APs into COPII vesicles requires specific lipid interactions. We compared the ER exit mechanism and segregation of GPI-APs from other secretory proteins in mammalian and yeast cells. We find that, unlike yeast, ER-to-Golgi transport of GPI-APs in mammalian cells does not depend on sphingolipid synthesis. Whereas ER exit of GPI-APs is tightly dependent on Sar1 in mammalian cells, it is much less so in yeast. Furthermore, in mammalian cells, GPI-APs and other secretory proteins are not segregated upon COPII vesicle formation, in contrast to the remarkable segregation seen in yeast. These findings suggest that GPI-APs use different mechanisms to concentrate in COPII vesicles in the two organisms, and the difference might explain their propensity to segregate from other secretory proteins upon ER exit.