Male sterility is an important contributor to heterosis in Brassica napus L. The B. napus line 7-7365ABC is a recessive epistatic genic male sterile (REGMS) three-line system. The 7-7365A line with the genotype Bnms3ms3ms4ms4RfRf is male-sterile, while the 7-7365B line with the genotype BnMs3ms3ms4ms4RfRf is male-fertile, and 7-7365C with homozygous recessive genotypes at the three loci shows male fertility because the loss function of Bnrf gene causes the inhibition of the genetic trait of the double mutant Bnms3 Bnms4. Histological studies addressing male sterility, transcriptional regulation pathways and the role of abscisic acid (ABA) in the anther development of REGMS plants are reported here. In the male-sterile line 7-7365A, tapetum cell and microspore mother cell separation were affected, and this led to failure of microspore release. The activity of polygalacturonase and the expression of the pectin methylesterase gene (AT3g06830) were significantly downregulated. Nine genes were downregulated in 7-7365A compared to 7-7365B and 7-7365C, including genes specifically expressed in tapetum (A3, A9, MS1) and the ABA-responsive gene KIN1. ABA concentration in 7-7365B was significantly higher than in 7-7365A and 7-7365C in young flower buds. Furthermore, temperature treatment made some sterile 7-7365A flowers become fertile. The stamens in these flowers produced viable pollen, and filament elongation was restored to its level in 7-7365C. We propose that ABA might control the expression of genes involved in cell separation during early anther development. The REGMS phenotype could be controlled by a primary pathway of male sterile metabolism positively regulated by the BnMs3 gene and a supplementary pathway negatively regulated by the BnRf gene.