We identified a clinical patient with a biallelic mutation in CETN3 exhibiting symptoms akin to microcephaly. This discovery prompted our investigation into the role of CETN3 in neurodevelopment. To delve deeper, we established a CETN3-KO H9 cell line and cultivated cerebral organoids using it. Mirroring the patient's phenotype, our cerebral organoids exhibited smaller sizes compared to the control group. Through immunofluorescence analysis, we observed that CETN3 deficiency interfered with neurodifferentiation and diminished the proliferation of neural stem cells. Additionally, our findings indicated that CETN3 knockout led to abnormal division of neural stem cells by impacting centrosome function.