Mature pollen is very sensitive to cold stress in chilling-sensitive plants. To understand the genetic regulation of tolerance to cold stress, we analyzed the transcript expression profile in mature pollen of Arabidopsis using Affymetrix GeneChips containing ~24,000 genes. Expression of 2,127 genes was cold-regulated, of which 697 genes were upregulated and 1,430 genes were downregulated. Further analysis showed that a large number of signal transduction components were significantly affected by cold treatment, indicating extensive changes in the gene regulatory networks of mature pollen. Many cold-responsive genes encode transcription factors, suggesting a multitude of transcriptional cascades. A number of genes important for the biosynthesis or signaling of plant hormones, such as abscisic acid, auxin, and jasmonate, were regulated by cold stress, which is of potential importance in coordinating cold tolerance with pollen growth and development. In addition, 159 mature pollen-specific genes that might be involved in pollen viability were also cold-regulated. Expression of the cold-responsive transcripts identified by microarray analysis was confirmed by quantitative real-time PCR. Our study provides an overall picture of the cold-responsive transcriptome in Arabidopsis pollen and is valuable for understanding gene regulation in response to cold stress and the molecular mechanisms of cold tolerance in mature pollen.