Background Istradefylline (IST), an adenosine A2A receptor antagonist, has been used since 2013 in Japan as an adjunctive therapy to levodopa (L‐DOPA) for patients with Parkinson's disease (PD). The long‐term use of IST as an adjunct to L‐DOPA (IST‐LD) was herein investigated to clarify the cooperative potential to keep motor functions, and an epigenetic modification for disease‐specific up‐regulated A2AR signals. Methods The cohort comprising 62 PD patients with diurnal variations in motor function were treated with IST‐LD for 36 months, and clinical and biomarker parameters were evaluated. One monozygotic twin pair with juvenile PD and eight healthy control (HC) subjects were recruited for analyses of epigenetic biomarkers with peripheral blood lymphocyte (PBL): the A2AR protein (A2AR‐p), A2AR mRNA (A2AR‐m), and DNA methylation of the ADORA2A (Chr22q11.23) and DRD1 (Chr5q35.1) genes. Results IST‐LD partially reversed locomotive dysfunction and motor off time and did not promote the severe dyskinesia (LID) develop. A2AR expression levels of the PBL were higher in IST‐naïve PD patients than in HC, which were associated with the effectiveness of IST‐LD and clinical global impression improvements. Intrinsic DNA demethylation of the ADORA2A gene were observed in juvenile monozygotic twin pair of the PD and IST‐naïve PD patients, which were reversed by IST‐LD at 12 months. Conclusions IST‐LD was cooperative for long term to preserve motor execution controls and global daily activities improvement, through the canonical pharmacodynamics of inhibiting A2AR signaling, and also via the epigenetic modification on the ADORA2A gene.