The key gluconeogenic enzyme fructose-1,6-bisphosphatase (FBPase) is subjected to catabolite inactivation and degradation when glucose-starved cells are replenished with fresh glucose. In various studies, the proteasome and the vacuole have each been reported to be the major site of FBPase degradation. Because different growth conditions were used in these studies, we examined whether variations in growth conditions could alter the site of FBPase degradation. Here, we demonstrated that FBPase was degraded outside the vacuole (most likely in the proteasome), when glucose was added to cells that were grown in low glucose media for a short period of time. By contrast, cells that were grown in the same low glucose media for longer periods of time degraded FBPase in the vacuole in response to glucose. Another gluconeogenic enzyme malate dehydrogenase (MDH2) showed the same degradation characteristics as FBPase in that the short term starvation of cells led to a non-vacuolar degradation, whereas long term starvation resulted in the vacuolar degradation of this protein. The N-terminal proline is required for the degradation of FBPase and MDH2 for both the vacuolar and non-vacuolar proteolytic pathways. The cAMP signaling pathway and the phosphorylation of glucose were needed for the vacuolar-dependent degradation of FBPase and MDH2. By contrast, the cAMP-dependent signaling pathway was not involved in the non-vacuolar degradation of these proteins, although the phosphorylation of glucose was required.