Objective This study was performed to explore the expression of α-smooth muscle actin (α-SMA) in the periodontal ligament (PDL) of young and adult rats during post-emergent tooth eruption in opposed and unopposed teeth at two time points: 3 and 15 days after antagonist loss. Methods Four-week-old (n = 20) and 22-week-old (n = 20) male Wistar rats were used. The right maxillary molar crowns were cut down. PDL samples were isolated from the first mandibular molars at two time points: 3 and 15 days after cut-down of the right maxillary molars. Quantitative reverse-transcription polymerase chain reaction and immunohistochemical staining were performed to detect differences in α-SMA expression in the PDL tissues of unopposed versus opposed molars. Results α-SMA was upregulated in the PDL of the unopposed molars in the 3-day group of young rats. The region around the root apex of the unopposed molars in this group exhibited strong immunostaining for α-SMA. The expression level and immunoreactivity of α-SMA did not differ in both time points in young controls and among all the adult groups. Conclusion α-SMA-positive myofibroblasts are implicated in post-emergent tooth eruption of unopposed molars of young animals.