Brain specific kinases (BRSKs) are serine/threonine kinases, preferentially expressed in the brain after Embryonic Day 12. Although BRSKs are crucial neuronal development factors and regulation of their enzymatic activity has been widely explored, little is known of their transcriptional regulation. In this work, we show that Neuronal Growth Factor (NGF) increased the expression of Brsk1 in PC12 cells. Furthermore, during neuronal differentiation, Brsk1 mRNA increased through a MAPK-dependent Sp1 activation. To gain further insight into this regulation, we analyzed the transcriptional activity of the Brsk1 promoter in PC12 cells treated with NGF. Initially, we defined the minimal promoter region (−342 to +125 bp) responsive to NGF treatment. This region had multiple Sp1 binding sites, one of which was within a CpG island. In vitro binding assays showed that NGF-induced differentiation increased Sp1 binding to this site and that DNA methylation inhibited Sp1 binding. In vitro methylation of the Brsk1 promoter reduced its transcriptional activity and impaired the NGF effect. To evaluate the participation of DNA methyltransferases in Brsk1 gene regulation, the 5′Aza-dC inhibitor was used. 5′Aza-dC acted synergistically with NGF to promote Brsk1 promoter activity. Accordingly, DNMT3B overexpression abolished the response of the Brsk1 promoter to NGF. Surprisingly, we found Dnmt3b to be a direct target of NGF regulation, via the MAPK pathway. In conclusion, our results provide evidence of a novel mechanism of Brsk1 transcriptional regulation changing the promoter's methylation status, which was incited by the NGF-induced neuronal differentiation process.