Background: Transcription factor Pax6 plays an essential role in the expression of other transcription factors, cell adhesion molecules and is crucial for neurogenesis in the developing forebrain. Analysis of gene expression profiles through microarray experiments in Pax6 mutants allowed us to focus on CRALBP, one of the many genes that were downregulated. Methods: We studied the expression of CRALBP in wt and Pax6-/- mutants through in situ hybridization and immunohistochemistry. ChIP assay and luciferase reporter assay were performed to show the regulatory role of Pax6 on CRALBP promoter. Results: RNA and protein expression data show that CRALBP expression was completely abolished in Pax6 mutants. In vivo binding assays and in vitro reporter assays indicate that Pax6 not only binds the promoter of CRALBP but also positively regulates protein expression. Conclusions: This work provides evidence supporting that CRALBP is a direct downstream target of Pax6. However, the role of CRALBP in the cortex is yet to be elucidated. General Significance: Pax6 is a marker expressed on neural stem cells and progenitor cells. Understanding Pax6-dependent gene regulatory mechanisms unravels signaling cascades that occur early during development. © 2011 Elsevier B.V. All Rights Reserved.