Introduction: The aim of the study was to compare the number of paired box 7 (PAX7)-positive, myogenic differentiation 1 (MyoD1)-positive, and neural cell adhesion molecule-1 (NCAM)-positive satellite cells in human primary versus secondary inferior oblique muscle overaction (IOOA). Methods: This prospective observational study enrolled patients who underwent inferior oblique muscle myectomy at the Department of Pediatric Ophthalmology and Strabismus in Tianjin Eye Hospital between January 2020 and November 2020. The muscle specimens were processed. Immunohistochemistry and immunofluorescence were used to quantify inferior oblique muscle fiber diameter and PAX7-positive, NCAM-positive, and MyoD1-positive satellite cells. Results: Thirty-eight patients with inferior oblique overaction were enrolled: 18 with primary IOOA and 20 with secondary IOOA. The participants were significantly younger in the secondary IOOA group than in the primary IOOA group (2.8 ± 1.2 vs. 9.0 ± 3.2 years, p < 0.001). The muscle fiber diameter between the two groups was not significantly different (13.5 ± 1.4 vs. 13.8 ± 0.7 µm, p = 0.530), but PAX7+ (3.3 ± 2.8 vs. 1.8 ± 0.6, p < 0.001), NCAM+ (3.6 ± 1.5 vs. 1.7 ± 0.2, p < 0.001), and MyoD1+ (4.8 ± 1.9 vs. 2.7 ± 0.5, p < 0.001) cell counts were higher in primary IOOA than in secondary IOOA. Conclusions: PAX7pos, MyoD-1pos, and NCAMpos cell counts per laminar nucleus were almost 2-fold higher in the primary IOOA group than in the secondary IOOA group.