BACKGROUND: COVID-19 characterized by refractory hypoxemia increases patient mortality because of immunosuppression effects. This study aimed to evaluate the efficacy of immunomodulatory with thymosin alpha1 for critical COVID-19 patients. METHODS: This multicenter retrospective cohort study was performed in 8 government-designated treatment centers for COVID-19 patients in China from Dec. 2019 to Mar. 2020. Thymosin alpha1 was administrated with 1.6 mg qd or q12 h for >5 days. The primary outcomes were the 28-day and 60-day mortality, the secondary outcomes were hospital length of stay and the total duration of the disease. Subgroup analysis was carried out according to clinical classification. RESULTS: Of the 334 enrolled COVID-19 patients, 42 (12.6%) died within 28 days, and 55 (16.5%) died within 60 days of hospitalization. There was a significant difference in the 28-day mortality between the thymosin alpha1 and non-thymosin alpha1-treated groups in adjusted model (P = 0.016), without obvious differences in the 60-day mortality and survival time in the overall cohort (P > 0.05). In the subgroup analysis, it was found that thymosin alpha1 therapy significantly reduced 28-day mortality (Hazards Ratios HR, 0.11, 95% confidence interval CI 0.02-0.63, P=0.013) via improvement of Pa02/FiO2 (P = 0.036) and prolonged the hospital length of stay (P = 0.024) as well as the total duration of the disease (P=0.001) in the critical type patients, especially those aged over 64 years, with white blood cell >6.8x10(9)/L, neutrophil >5.3x10(9)/L, lymphocyte < 0.73 x 10(9)/L, PaO2/FiO2 < 196, SOFA > 3, and acute physiology and chronic health evaluation (APACHE) II > 7. CONCLUSION: These results suggest that treatment with thymosin alpha1 can markedly decrease 28-day mortality and attenuate acute lung injury in critical type COVID-19 patients.