Comparative expression profiling of flower buds in two male sterile lines [genic male sterile (GMS) and cytoplasmic male sterile (CMS)] with its male fertile maintainer line in Chinese cabbage pak-choi was performed using cDNA-AFLP technology to identify the genes implicated in male sterility. A novel gene BcMF10, sharing high sequence similarity to the function-unknown DUF1216 family in Arabidopsis was isolated, whose expression was absent in the flower buds of the GMS and CMS lines but present in the male fertile maintainer line. Temporal and spatial expression pattern analysis revealed that BcMF10 began to be expressed in tapetal cells and microspores during meiosis. Expression in tapetal cells was persistent until the degeneration of tapetum, and expression in microspores reached a peak during the tetrad stage but gradually declined as development proceeded. RNA interference technology was used to address the biological function of BcMF10. The RNAi transgenic Chinese cabbage pak-choi lines showed normal vegetative growth and reproductive development, but poor pollen germination. Scanning electron microscopy (SEM) showed that most of the transgenic pollen was deformed and exhibited an irregular shape with an abnormal number and distribution of germinal furrows. It is speculated that BcMF10 may encode a protein that plays a role in the formation of intine wall.