In this report a full length cDNA, Brassica campestris Male Fertile 21 (BcMF21) was successfully isolated from one of the cDNA-amplified fragment length polymorphism (cDNA-AFLP) transcript-derived fragments (TDFs), BBP10, that was found down-regulated in the flower buds of sterile plants in Brassica campestris L. ssp. chinensis Makino genic male sterile (GMS) AB line system (Bcajh 97-01A/B). BcMF21 protein structure analysis showed a signal peptide at the N-terminus; two protein kinase C phosphorylation sites, five N-myristoylation sites and one casein kinase II phosphorylation site. The promoter region of BcMF21, a 779 bp upstream of ATG was isolated by thermal asymmetric interlaced-PCR (TAIL-PCR). Bioinformatics analysis revealed that the promoter of BcMF21 contained several classical cis-acting elements and three pollen specific elements. Transient expression analysis showed that the promoter could drive green fluorescence protein (GFP) expression. Quantitative reverse transcript-PCR analysis revealed that BcMF21 was specifically expressed in flower buds. The transcript level of BcMF21 was much lower in the sterile flower buds than in the fertile flower buds in 'Bcajh 97-01A/B' system. In situ hybridization further showed that BcMF21 was only expressed in the tetrads and the microspores at the tetrad stage and the uninucleate stage. In addition, phylogenetic analysis demonstrated that the BcMF21 was relative conserved within family Crucifereae and might be originated from the ancestor diploid B. campestris within genus Brassica according to the Triangle of U theory.