To reveal the possible molecular mechanism underpinning the male sterility induced by a sulfonyflurea herbicide tribenuron-methyl (TBM), a comparative transcriptome analysis between rapeseed plants exposed to a TBM solution of 0.2 mg/L concentration and the control (spray with water) was conducted by using Illumina digital gene expression tag profiling technology (DGE). A transcriptomic analysis of the flower buds of TBM treatment identified 208 up-regulated and 164 down-regulated differential expression genes in comparison with the control. It was showed by the genetic network that the main groups of GO terms including response to chemical and hormone stimulus (response to stress), developmental process (pollen wall and pollen exine formation), lipid metabolism, and cellular amino acid derivative biosynthesis process were significantly influenced by the TBM treatment. The cytological and transcriptomic alterations in the treated plants were in accordance with reduction of chlorophyll, photosynthetic rate, and elevation of ethylene release. Overall design: The young flower buds (microsporocyte cell (MMC) to uni-nucleate microspore stage, length = 3 mm) corresponding to the key stage sensitive to gametocide, were dissected from the plants 5 days after treatment and the control plants. The two biological replicates were designed as HS1, HS2 for the treatment and CK1 and CK2 for the control.Two groups of Illumina DGE (digital gene-expression tag profiling) libraries (HS1 and HS2) and the control (CK1 and CK2) were constructed using the cDNAs of young floral buds of TBM-treated plants and the control with Illumina's DGE Tag Profiling Kit according to the manufacturer's protocol. We performed a single end sequencing on an Illumina Hiseq2500 platform following the vendor's recommended protocol. The raw data containing adaptor sequences, tags with low quality sequences and unknown nucleotides N were filtered to obtain clean reads with 36 nt in length. Clean reads were then conducted for quality assessment. All clean tags were aligned to the Brassica genome in JGI (http://genome.jgi.doe.gov/) by Bowtie2 ver 2.1.0 (http://sourceforge.net/projects/bowtie-bio/files/bowtie2/2.1.0/), only 1 bp mismatch is allowed. The number of perfect reads matching to each unigene was normalized to Transcripts Per Kilobase of exon model per Million mapped reads (TPM). The significant DETs among the two groups of samples were selected, with false discovery rate (FDR) = 0.05 and fold-change = 2. The sequence of each DET was further searched by BLAST to get more information in NCBI nucleotide collection database and the functional annotation was searched in Uniprot databse (http://www.uniprot.org/). The genetic networks of GO (Gene Ontology) terms for the DETs were drew by BiNGO software in Cytoscape (www.cytoscape.org/).