Malignant gliomas (MG) are highly infiltrative tumours with a poor prognosis. Nuclear factor I (NFI) is a family of 4 transcription factors (NFIA, B, C and X) implicated in the regulation of genes involved in MG cell migration and infiltration, particularly the neural stem cell marker, brain fatty acid binding protein (B-FABP). NFI activity is regulated by its phosphorylation status, with hypophosphorylated NFI being the active form. Our results indicate that the phosphatase calcineurin is able to dephosphorylate NFI. In turn, calcineurin is cleaved and activated by calpain proteases. We have identified CAST, a gene that encodes calpain inhibitor, calpastatin, as a putative target of NFI based on chromatin immunoprecipitation. Putative NFI binding elements are located in intron 3 of the CAST gene. To determine whether there is a bona fide alternative promoter within intron 3 of CAST, we carried out gel shifts as well as luciferase reporter gene assays using both the canonical and alternative promoters of CAST. These assays confirmed CAST alternative promoter usage in MG cells. Knockdown of individual NFIs revealed a role for NFIC and NFIX in the repression of CAST gene expression, specifically in cells expressing the hypophosphorylated (active) form of NFI. NFI depletion also altered the subcellular localization of both calpain and calineurin protein. Our results suggest a feedback loop for the NFI –calcineurin – calpain – calpastatin pathway in MG cells which may regulate cell migration.