As one of the largest families of transcription factors in plants, the R2R3-MYB proteins play important roles in diverse biological processes including growth and development, primary and secondary metabolism such as flavonoid and anthocyanin biosynthesis as well as abiotic and biotic stress responses. However, functions of R2R3-MYB genes in rapeseed (Brassica napus L.) remain elusive. Here, we characterized BnaMYB111L, which is homologous to Arabidopsis MYB111 and encodes an R2R3-MYB protein in rapeseed. BnaMYB111L is responsive to abscisic acid (ABA), heat, cold, hydrogen peroxide and fungal pathogen Sclerotinia scelerotiorum treatments through quantitative RT-PCR assay. BnaMYB111L encodes a transcriptional activator and is localized exclusively to nuclei. Interestingly, overexpression of BnaMYB111L in tobacco (Nicotiana benthamiana) and rapeseed protoplasts promoted reactive oxygen species (ROS) production and hypersensitive response-like cell death, accumulation of malondialdehyde (MDA) as well as degradation of chlorophyll. Furthermore, BnaMYB111L expression evoked the alterations of transcript levels of genes encoding ROS-producing enzyme, vacuolar processing enzymes and genes implicated in defense responses. A further dual luciferase reporter assay indicated that BnaMYB111L activated the expression of RbohB, MC4 and ACRE132, which are involved in ROS generation, cell death as well as defense responses. Taken together, this study characterized the function of rapeseed MYB111L and identified its putative target genes involved in ROS production and cell death.