Type 1 narcolepsy is strongly (98%) associated with human leukocyte antigen (HLA) class II DQA1*01:02/DQB1*06:02 (DQ0602) and highly associated with T cell receptor (TCR) alpha locus polymorphism as well as other immune regulatory loci. Increased incidence of narcolepsy was detected following the 2009 H1N1 pandemic and linked to Pandemrix vaccination, strongly supporting that narcolepsy is an autoimmune disorder. Although recent results suggest CD4+ T cell reactivity to neuropeptide hypocretin/orexin and cross-reactive flu peptide is involved, identification of other autoantigens has remained elusive. Here we study whether autoimmunity directed against Regulatory Factor X4 (RFX4), a protein co-localized with hypocretin, is involved in some cases of narcolepsy. Studying human serum, we found that autoantibodies against RFX4 were rare. Using RFX4 peptides bound to DQ0602 tetramers, antigen RFX4-86, -95, and -60 specific human CD4+ T cells were detected in 4/10 patients and 2 unaffected siblings, but not in others. Following culture with each cognate peptide, enriched autoreactive TCRαβ clones were isolated by single-cell sorting and TCR sequenced. Homologous clones bearing TRBV4-2 and recognizing RFX4-86 in patients and one twin control of patient were identified. These results suggest the involvement of RFX4 CD4+ T cell autoreactivity in some cases of narcolepsy, but also in healthy donors.