Cytoplasmic male sterility is primarily caused by chimeric genes located in the mitochondrial genomes. Previously, orf288 had been cloned as a CMS-associated gene in the hau CMS line. However, neither the specific abortive stage nor the molecular function of orf288 had been determined in hau CMS. To better understand the mechanism of hau CMS in Brassica, we further characterized the hau CMS line and found defective mitochondrial arrest during the development of archesporial cells during the L2 stage, leading to male sterility. Expression level of the orf288 transcript was higher in the male sterility line than in the restorer line, although no significant differences were apparent at the protein level. The toxicity region of ORF288 is located near the N-terminus and represses E. coli growth. However, transgenic expression of different portions of ORF288 indicated that the region that causes male sterility resides between amino acids 73 and 288, which do not result in growth inhibition when expressed in E. coli. Transcriptome analysis revealed a wide range of genes involved in anther development and mitochondrial function that were differentially expressed in the hau CMS line. This study provides new insights into hau CMS mechanism by which orf288 affects Brassica juncea fertility.