Graphene oxide (GO) is an oxidized form of graphene that has the potential to be used as a nanomedicine for the treatment of neurodegenerative and other physiological disorders. We have previously demonstrated that GO exhibits minimal endocrine-disrupting effects in Japanese medaka (Oryzias latipes) due to physical aggregation and accumulation rather than direct endocrine interference. Moreover, males are more sensitive to GO than females. We have extended our investigations to the nervous system of the fish, concentrated on the cerebellum (CB), and considered Purkinje cells (PKCs), which play a significant role in motor control, as a potential target of GO. The experimental adult fish were exposed to GO in vivo by immersion (IMR) (20 mg/L in BSS) and by a single intraperitoneal (IP) injection (100 µg/g). Parallel controls were maintained either in BSS (IMR) or injected with nanopure water (IP). An evaluation of the CB was made in paraffin sections after haematoxylin/eosin (H&E) staining. The CB is located dorsally to the midbrain and caudally to the optic tectum and primarily consists of three lobular structures, the valvula cerebelli (VC), the corpus cerebelli (CC), and the lobus caudalis cerebelli (LC). The PKCs appear to be significantly larger with a droplet-like shape, prominent nucleus and nucleolus and are distributed between the granular and molecular layers in VC and CC, not in LC. We assessed the histomorphology, distribution/density, nuclear (N) and perikaryal (S) areas, and (N:S) ratio of the PKCs found in the CC area of the CB. We observed that females have apparently larger PKCs than males and GO exposure altered cell morphology and morphometry and reduced the distribution of PKCs, which was dependent on the exposure route and sex of the fish. Further studies are needed to elucidate the mechanistic basis of GO-induced neurotoxicity before its application as a nanomedicine for the treatment of various neurological disorders.