Microcephaly is a disease in which the head circumference and brain volume decrease. Patients with microcephaly show a range of cognitive deficits. One of the causes of microcephaly is mutations in the RTTN gene. RTTN mutations are responsible for syndromic malformations of cortical development. Here we have generated on-chip human brain organoids and 3D-hippocampal organoids to study the role of RTTN in the developing human brain.A RTTN homozygous pathogenic mutation was introduced in human embryonic stem cells using CRISPR/Cas9 genome editing. The developing control on-chip brain organoids exhibit hallmarks of a well-organized neuroepithelium composed of proliferating radial glia, showing interkinetic nuclear motility and undergoing mitosis close to the lumen. However, the mutant ones revealed a significant growth impairment. Analysis of time-lapse live-cell images revealed that the mutant progenitors' mitosis duration was 2.4-fold slower than control ones (p<0.0001). Interkinetic nuclear motility, particularly movement towards the basal side, was significantly slower in the nuclei of the mutated RTTN group (p<0.01). Mutant-derived hippocampal organoids exhibited impaired growth over fifty days (p<0.0001). Sixty-four-day-old mutant hippocampal organoids exhibited more cycling cells and increased phospho-histone-H3 positive cells, suggesting a longer duration of the G2-M phase of the cell cycle. In addition, increased apoptosis was observed in mutant hippocampal organoids. We showed that a RTTN pathogenic mutation results in a microcephaly-like phenotype of human brain organoids. The cellular defects of RTTN-related microcephaly included prolonged mitosis, changes in interkinetic nuclear migration, and increased apoptosis. These results suggest that RTTN has an important role during human brain development.