||Background: Pollen, the male partner in the reproduction of flowering plants, comprises either two or three cells at maturity. The current knowledge of the pollen transcriptome is limited to the model plant Arabidopsis thaliana, which has tri-cellular pollen grains at maturity. Comparative studies on pollen of other genera, particularly crop plants, are needed to understand the pollen gene networks that are subject to functional and evolutionary conservation. In this study, we used the Affymetrix Soybean GeneChip to perform transcriptional profiling on mature bi-cellular soybean pollen. Results: Compared to the sporophyte transcriptome, the soybean pollen transcriptome revealed a restricted and unique repertoire of genes, with a significantly greater proportion of specifically expressed genes than is found in the sporophyte tissue. Comparative analysis shows that, among the 37,500 soybean unique transcripts addressed in this study, 10,299 genes (27.46%) are expressed in pollen. Of the pollen-expressed genes, about 9,489 (92.13%) are also expressed in sporophytic tissues, and 810 (7.87%) are selectively expressed in pollen. Overall, the soybean pollen transcriptome shows an enrichment of transcription factors (mostly zinc finger family proteins), cell cycle-related transcripts, signal recognition receptors, ethylene responsive factors, chromatin remodeling factors, and members of the ubiquitin proteasome proteolytic pathway. Moreover, we identify several new pollen-specific candidate genes that might play a significant role in pollen biology. Conclusion: This is the first report of a soybean pollen transcriptional profile. These data extend our current knowledge regarding regulatory pathways that govern the gene regulation and development of pollen. We also demonstrate that pollen is a rich store of regulatory proteins that are essential and sufficient for de novo gene expression. A comparison between transcription factors up-regulated in soybean and those upregulated in Arabidopsis revealed some divergence in the numbers and kinds of regulatory proteins expressed in both species.