Astrocyte is a type of glial cell involved in synaptic transmission and the formation and maturation of synapses. Currently, the establishment of induced pluripotent stem cells (iPSCs) allows the differentiation of stem cells into various types of cells while preserving the patient phenotype. Therefore, patient iPSCs replace animal models in pathological analysis and drug discovery. Technological advances have provided access to human iPSC-derived astrocytes (HiAs). Furthermore, neurons co-cultured with pathological astrocytes have been used to study their morphology, protein levels, and spontaneous synaptic responses. However, these studies did not investigate detailed synaptic functions such as synaptic transmission evoked by electrical stimulation and morphological analysis at the single neuron level. In this study, we established autaptic cultures with HiAs (HiAs Autaptic Cultures, HiAACs), single neuron cultures grown in isolation on microislands of HiAs that form synapses exclusively with themselves. We found that neurons in HiAACs develop morphologically by co-culture with HiAs and form functional synapses that exhibit excitatory postsynaptic currents. Although we used healthy astrocytes in this study, HiAACs can be used to study various diseases by using patient-derived astrocytes in the future.This work was supported by funding from JSPS, AMED, MEXT, the Science Research Promotion Fund and The Fukuoka University Fund, and Kyushu University Hospital.