The Onecut (OC) family of transcription factors comprises three members in mammals, namely HNF-6 (or OC-1), OC-2 and OC-3. During embryonic development, these transcriptional activators control cell differentiation in pancreas, in liver and in the nervous system. Adult Hnf6 mutant mice exhibit locomotion defects characterized by hindlimb muscle weakness, abnormal gait and defective balance and coordination. Indeed, HNF-6 is required in spinal motor neurons for proper formation of the hindlimb neuromuscular junctions, which likely explain muscle weakness observed in corresponding mutant animals. The goal of the present study was to determine the cause of the balance and coordination defects in Hnf6 mutant mice. Coordination and balance deficits were quantified by rotarod and runway tests. Hnf6 mutant animals showed an increase in the fall frequency from the beam and were unable to stay on the rotarod even at low speed, indicating a severe balance and coordination deficit. To identify the origin of this abnormality, we assessed whether the development of the main CNS structure involved in the control of balance and coordination, namely the cerebellum, was affected by the absence of HNF-6. Firstly, we observed that Hnf6 was expressed transiently during the first week after birth in the Purkinje cells of wild type newborn mice. Secondly, we showed that, in Hnf6−/− mice, the organization of Purkinje cells became abnormal during a second phase of their development. Indeed, Purkinje cells were produced normally but part of them failed to reorganize as a regular continuous monolayer at the interface between the molecular and the granular layer of the cerebellum. Thus, the Onecut factor HNF-6 contributes to the reorganization of Purkinje cells during a late phase of cerebellar development.