PURPOSE: This study investigated the long-term efficacy and safety of Autologous Platelet Concentrate (APC) injection as an adjunct to pars plana vitrectomy (PPV) in the treatment of macular holes (MHs) with a high risk of surgical failure, including large, highly myopic, or recurrent MHs. STUDY DESIGN: Retrospective study. METHODS: This study included patients who underwent PPV with or without APC injection for MHs at Seoul St. Mary's Hospital between March 2017 and February 2020, with a minimum postoperative follow-up period of 3 years. Anatomical success rates, alterations in foveal microstructure, and improvements in best-corrected visual acuity (BCVA) were evaluated. RESULTS: Fifty-four patients were included, exhibiting a trend toward enhanced anatomical success in the PPV with APC group (95.0%) compared to the PPV with flap techniques (85.7%) and PPV without the adjunct of flap techniques or APC injection (84.6%), though not statistically significant. Significant findings included increased glial proliferation within the PPV with APC group, sustained for up to one year (P <
0.01), and a significant correlation between glial proliferation and MH closure (P = 0.032). Improvements in BCVA were substantial and found to be significantly associated with preoperative BCVA, the reconstruction of the external limiting membrane (ELM) and ellipsoid zone (EZ) (P = 0.008, 0.010, and 0.047, respectively). CONCLUSION: The use of APC as adjuvant to PPV can be advantageous in cases of challenging MHs, reflecting comparable anatomical success rates, potential for glial proliferation critical for hole closure, and established safety profile.