Abstract Purpose HNF4A-maturity-onset diabetes of the young (MODY1) is a relatively rare subtype of monogenic diabetes caused by loss-of-function of the HNF4A gene, which encodes the transcription factor HNF4α. HNF4α is known to form heterodimers, and the various combinations of isoforms that make up these heterodimers have been reported to result in a diversity of targeted genes. However, the function of individual HNF4α variant isoforms and the heterodimers comprising both wild-type and variant HNF4α have not yet been assessed. Therefore, in this study, we analyzed the functional consequence of HNF4A D248Y variant in vitro. Methods We investigated the case of a 12-year-old Japanese girl who developed diabetes at 11 years of age. Genetic sequencing detected a novel heterozygous missense HNF4A variant (c.742G>T, p.Asp248Tyr; referred as “D248Y”) in the patient and her relatives who presented with diabetes. Results Although the wild-type HNF4α isoforms (HNF4α2, HNF4α3, HNF4α8, HNF4α9) enhanced the INS gene promoter activity in HepG2 cells, the promoter activity of D248Y was consistently low across all isoforms. The presence of D248Y in homodimers and heterodimers, comprising either HNF4α8 or HNF4α3 or a combination of both isoforms, also reduced the INS promoter activity in Panc-1 cells. Main Conclusions We report the clinical course of a patient with HNF4A-MODY and the functional analysis of novel HNF4A variants, with a focus on the isoforms and heterodimers they form. Our results serve to improve the understanding of the dominant-negative effects of pathogenic HNF4A variants.