In all metazoans, the expression of group B HMG domain Sox transcription factors is associated with the earliest stages of CNS development. In Drosophila, SoxNeuro (SoxN) is involved in dorso-ventral patterning of the neuroectoderm, and in the formation and segregation of neuroblasts. In this report, we show that SoxN expression persists in a subset of neurons and glial cells of the ventral nerve cord at embryonic stages 15/16. In an attempt to address SoxN function in late stages of CNS development, we have used a chromatin immunoprecipitation approach to isolate genomic regions bound in vivo by SoxN. We identified several genes involved in the regulation of axon scaffolding as potential direct target genes of SoxN, including beat1a, semaphorin2a, fasciclin2, longitudinal lacking and tailup/islet. We present genetic evidence for a direct involvement of SoxN in axonal patterning. Indeed, overexpressing a transcriptionally hyperactive mutated SoxN protein in neurons results in specific defects in axon scaffolding, which are also observed in transheterozygous combinations of SoxN null mutation and mutations in its target genes.