Background: Glia maturation factor beta (GMFβ) was initially purified as an extract from glioblastoma conditioned media that when applied to primary glioblastoma cultures, resulted in process extension in vitro. Subsequently, the cDNA was cloned, and it encoded a 17 kDa protein. Bioinformatic modelling predicted that GMFβ was a member of the actin depolymerising factor (ADF) family, most similar to cofilin. Objective: We sought to further characterise the role of GMFβ as an ADF promoting differentiation of neuroblastoma by modifying the actin cytoskeleton. Methods: We generated plasmids to over-express GMFβ and a mutant GMFβ with the deleted C-terminal predicted actin binding domain (GMFβ ΔABD). We also generated anti-peptide antibodies specific to an internal domain (GMFβP01) and to the predicted ABD (GMFβP02). Results: Treatment of cancer cells with cytochalasin D which disrupts the actin cytoskeleton significant modified the distribution of GMFβ within the cytoplasm, but colchicine, a microtubule inhibitor, had no effect. Immunolocalisation of GMFβ was in the axonal compartment of neurons in several regions of the human brain. Retinoic acid treatment of neuroblastoma cells resulted in neurite process extension with GMFβ redistribution, accordingly. Over-expression of GMFβ in neuroblastoma cells also promoted neurite extension, but not with the GMFβ ΔABD over-expressed. Knockdowns of GMFβ also reduced neurite extension. Of interest, GMFβ was expressed in the Schwannian stroma and ganglioneuroblasts, but not in undifferentiated neuroblastoma tumour cells. Conclusion: Targeting GMFβ expression may be a novel treatment strategy to promote neuroblastoma differentiation, perhaps as an adjunct to current isotretinoin administration following immunotherapy.