Background: Thymosin-\alpha1 has been implicated into the treatment of novel respiratory virus Coronavirus Disease 2019 (COVID-19), but the underlying mechanisms are still disputable. Aim: Herein we aimed to reveal a previously unrecognized mechanism that thymosin-\alpha1 prevents COVID-19 by binding with angiotensin-converting enzyme (ACE), which was inspired from the tool of network pharmacology. Methods: KEGG pathway enrichment of thymosin-\alpha1 treating COVID-19 was analyzed by Database of Functional Annotation Bioinformatics Microarray Analysis, then core targets were validated by ligand binding kinetics assay and fluorometric detection of ACE and ACE2 enzymatic activity. The production of angiotensin I, angiotensin II, angiotensin (1–7) and angiotensin (1–9) were detected by enzyme linked immunosorbent assay. Results: We found that thymosin-\alpha1 impaired the expressions of angiotensin-converting enzyme 2 and angiotensin (1–7) of human lung epithelial cells in a dose-dependent way (p 0.001). In contrast, thymosin-\alpha1 had no impact on their ACE and angiotensin (1–9) expressions but significantly inhibited the enzymatic activity of ACE (p > 0.05). Conclusion: The bioinformatic findings of network pharmacology and the corresponding pharmacological validations have revealed that thymosin-\alpha1 treatment could decrease ACE2 expression in human lung epithelial cells, which strengthens the potential clinical applications of thymosin-\alpha1 to prevent severe acute respiratory syndrome coronavirus 2 infection.