Plexin B3 is a neurally expressed transmembrane receptor protein participating in growth cone remodelling and synaptic plasticity. Despite their biological importance no plexin promoter has been characterized so far. Regulation of mouse plexin B3 transcription was investigated using in silico analysis of promoter and inititation area, 5′RACE, reporter-gene assays, gelshift and co-expression experiments. As a result we have described a novel 5′ exon and show that a 234 basepair region upstream of it exhibits promoter activity. Further analysis indicated that this region contains predicted binding sites for myeloid zinc finger protein 1 (MZF-1) and neurogenin 3 (Ngn3). Oligonucleotides corresponding to the recognition sequences of these factors produced a specific mobility shift in EMSA. Expression of the reporter gene attached to the 234 region was increased 2-fold by Ngn3 and reduced twice in response to MZF-1 over-expression. These results help to better comprehend mechanisms used for plexin B3 transcriptional regulation.