Thymosin β4 is a major actin sequestering protein in cells and can interact with G-actin. Thymosin β4 is found in many vertebrate tissues and cells and is especially concentrated in macrophages, fibroblasts, neutrophils, and platelets, which have large pools of G-actin. The main physiological role of Thymosin β4 is the regulation of actin polymerization. Thymosin β4 is also involved in angiogenesis, cell survival, cell migration and fetal development. Thymosin β4 plays a crucial role in the regulation of tight junction stability and acts in cytoskeleton rearrangement, which are closely related with BBB permeability. Thymosin β4 is a novel regulator for primary cilia formation and it affects ciliogenesis by regulating the expression of NPHP3 in HeLa cervical cancer cells. Thymosin β4 regulates HSC activation by influencing the activity of Smoothened and GLI2, suggesting Thymosin β4 as a novel therapeutic target in liver disease. Synthetic Thymosin β4 peptide increases NK cell cytotoxicity mediated by intercellular adhesion molecule-1 (ICAM-1 ) through the secretion of cytolytic granules to target cells, suggests that Thymosin β4 is a key activator of NK cell cytotoxicity. Thymosin β4 enhanced wound healing in a rat full thickness wound model suggest that Thymosin β4 is a potent wound healing factor with multiple activities. Thymosin β4 administration during gestation may act as a powerful fetal growth promoter, by accelerating the development of newborn organs and tissues.Recombinant Human Thymosin β4 significantly increased the survival rate of mice infected with MHV-A59 through inhibiting virus replication, balancing the host’s immune response, alleviating pathological damage, and promoting repair of the liver.