Objectives <p>To longitudinally follow up a cohort of patients with autotransplanted teeth, assessing how the timing of starting orthodontic treatment impacts root length.</p> Materials and methods <p>Patients under 18 with at least one open-apex autotransplanted premolar (AP) replaced to a central incisor position were included. Root/crown ratio (RCR) was calculated on periapical radiographs taken at intervals of 3–6 months after transplantation. The RCR of AP in patients without orthodontic treatment and patients where orthodontic treatment was initiated at different time points (3,6,9,12,18 months) were compared.</p> Results <p>52 autotransplanted premolars were included and 315 peri-apical radiographs were collected. The median timespan between autotransplantation and the start of orthodontic force (OF) application was 8.5 (IQR 2–23) months, with a mean follow-up period of 32 (IQR 9–46) months. Early OF post-autotransplantation correlated with early stagnation of root development. When OF was delayed, AP tended to show more root lengthening. Delayed OF was linked to a higher incidence of External Apical Root Resorption (EARR) once forces were introduced, ultimately resulting in root lengths comparable to those with early OF. The presence or absence of OF is the factor most notably influencing the final root length of AP.</p> Conclusions <p>The timing of OF application did not compromise the final root length of autotransplanted teeth. Early OF to prevent ankylosis is feasible without affecting root length. Future research should utilize 3D imaging to provide a deeper understanding of orthodontic treatment’s effects on autotransplanted teeth.</p> Clinical relevance <p>The study underscores the importance of tailored orthodontic intervention timing in autotransplanted teeth, balancing early force application to avoid ankylosis against the risk of EARR.</p>