In flowering plants, stamen development is a complex multistage process, which is highly regulated by a series of transcription factors. In this study, BcMF28, which encodes a R2R3-MYB transcription factor, was isolated from Brassica campestris. BcMF28 is localized in the nucleus and cytoplasm, and acts as a transcriptional activator. Quantitative real-time PCR and promoter activity analysis revealed that BcMF28 was predominately expressed in inflorescences. The expression of BcMF28 was specifically detected in tapetum, developing microspores, anther endothecium, and filaments during late stamen development. The overexpression of BcMF28 in Arabidopsis resulted in aberrant stamen development, including filament shortening, anther indehiscence, and pollen abortion. Detailed analysis of anther development in transgenic plants revealed that the degeneration of septum and stomium did not occur, and endothecium lignification was affected. Furthermore, the expression levels of genes involved in the phenylpropanoid metabolism pathway were altered in BcMF28-overexpressing transgenic plants. Our results suggest that BcMF28 plays an important regulatory role during late stamen development.