Objective <p>This study aimed to explore the role of miR-194 in the post-transcriptional regulation of PTPN12 and its impact on age-related thymic atrophy.</p> Methods <p>miR-194-5p and PTPN12 expression levels in thymus tissues from young and aged mice were assessed using quantitative PCR and western blot analysis. An aged-mouse model was established to overexpress varying levels of miR-194-5p. Expression levels of PTPN12, AKT, BAX, p50, and p65 in thymic epithelial cells were analyzed via quantitative PCR and western blot. Flow cytometry was used to evaluate TEC cell cycle progression, proliferation, and apoptosis. CD4⁺/CD8⁺ lymphocytes proportions in the spleen and peripheral blood were quantified, and structural changes in the thymus were examined using hematoxylin–eosin staining.</p> Results <p>miR-194 expression peaked in the thymus of middle-aged (6–7 months) C57BL/6 mice, while PTPN12 expression was highest in the young (1–2 months) mice and declined with age. TEC survival was enhanced in the miR-194-5p overexpression group, as indicated by cell cycle and apoptosis analyses. Additionally, CD4⁺ and CD8⁺ T cell counts and proportions in the spleen and peripheral blood were reduced in the miR-194-5p overexpression group. Compared to controls, the high-dose miR-194-5p group exhibited significantly increased p65 and p50 expression, while PTPN12 and BAX levels were markedly reduced, but AKT level was markedly increased.</p> Conclusion <p>This study demonstrated that miR-194-5p modulates thymic immunological function in aged mice, highlighting its potential role in age-related thymic atrophy.</p>