The pseudobulb of Oncidium orchid plays a key role in water, carbohydrate, and other nutrition support during floral development, yet a large scale of gene expression analysis involved in the metabolisms have not been evaluated. By subtracting RsaI-digested cDNAs of leaf from those of psuedobulb, an efficient subtractive cDNA library was developed. In total, 1080 subtractive expressed sequence tags (ESTs) were obtained. Analysis revealed approximately 636 unique gene parts, 120 clusters and 516 singles. Of these sequences, 74.8% were annotated on the database of NCBI GenBank. Peroxidase, sodium/dicarboxylate cotransporter, and mannose-binding lectin were highly expressed. Some gene profiles were identified as related to carbohydrate metabolism involved in mannan, pectin, starch and sucrose biosynthesis. A large fraction of the ESTs (35%) were classified into transportation, stress-related, cell cycle, or regulatory functions. Most genes that were differentially expressed are important in early flowering development, carbohydrate metabolism and stress-response physiology. This efficient organ-specific EST library represented an explicit transcriptome profile of Oncidium pseudobulb.