Transcription factor Pax6 plays an important role in fate determination of neural progenitor cells in animal models, yet, its distribution and role in the human developing brain have not been reported. Here we demonstrated that Pax6 was strongly expressed in dorsal and ventral proliferative zones, mainly in proliferating radial glia (RG) cells, some neuronal and intermediate progenitors, and sporadic deep cortical plate neurons. In contrast to reports in rodents, Pax6 in the human fetal brain occasionally colocalized with ventral transcription factor Olig2 in progenitor cells. Transfection with short interfering RNA abolished Pax6 expression in the cell cultures of human fetal RG, and significantly decreased the number of neurons generated from Pax6 knock-down cells. Hence, Pax6 has a critical role in neurogenic regulation of RG cells in the human forebrain, similar to reports in rodents. What is different in human forebrain is that Pax6 seems to regulate not only the genesis of cortical pyramidal neurons, but also a subpopulation of interneurons from both dorsal and ventral sources. Thus, regional distribution, colocalization with Olig2, and the role of Pax6 in neurogenesis of both projection and interneurons, suggest that developmental regulation by transcription factors may differ in primates and nonprimate mammals.