Background Relapse after orthodontic tooth movement (OTM) is frequently observed, and adjunctive therapy during the retention phase is often required to prevent it. Photobiomodulation therapy (PBMT) may be used as an adjunct to prevent relapse by stimulating osteogenic molecular pathways. To evaluate relapse prevention during the retention phase using red diode laser PBMT (650 nm, energy density 4 J/cm², 20 seconds per buccal and palatal region) applied to the tension side distal to the left maxillary first molar in Rattus norvegicus (Wistar strain), at a distance of 1 cm. Outcomes included the expression of BMP2, RUNX2, ALP, Type I Collagen, woven bone formation, and morphometric measurement of the M1–M2 interdental distance. Methods A true experimental study with a randomized post-test only control group design was conducted on 64 male Wistar rats. OTM was induced using a 10 g closed-coil spring for 14 days. Subsequently, the orthodontic force was removed (retention phase), and observations were made from day 15 to day 21 (7 days) and day 15 to day 28 (14 days). The subjects were divided into four groups: no treatment (K–), retainer without PBMT (K+1), PBMT without retainer (K+2), and combined retainer and PBMT (KP). Statistical analyses included Shapiro–Wilk test, Levene’s test, ANOVA, Tukey HSD post hoc test, and Pearson correlation. Evaluations were performed using immunohistochemistry (BMP2, RUNX2, ALP, Type I Collagen), hematoxylin–eosin staining (woven bone), and morphometric measurement of M1–M2 distance. Results The expression of all osteogenic biomarkers and woven bone formation increased significantly, following the pattern KP > K+2 > K+1 > K–, at both 7-day and 14-day retention periods (p < 0.001). There were significant effects of observation time, treatment, and their interaction on all osteogenic variables (p < 0.05). Biomarker expression levels at 14 days were higher than at 7 days. The K+2 group showed results comparable to KP, while KP demonstrated significantly greater effects than K+1. A significant negative Pearson correlation was observed between woven bone and M1–M2 distance (r = –0.59; p < 0.001). Conclusions PBMT at 650 nm shows potential as a non-invasive adjunctive therapy during the retention phase due to its ability to prevent post-OTM relapse through modulation of alveolar bone remodeling.