Thymosin beta belongs to the thymosin family， which consists of a series of highly conserved peptides involved in various biological processes. In teleosts， understanding of the immunological functions of thymosin beta is limited， particularly in vivo， which is essentially unknown. In the current study， we cloned and identified thymosin beta 4 from the teleost fish Golden pompano (Trachinotus ovatus)， which we have named TroTβ4. We investigated the expression patterns and functions of TroTβ4 in both in vivo and in vitro assays. TroTβ4 is composed of 44 amino acids and shares high sequence identities with known thymosin β4 species in other teleosts， which contains a highly conserved actin-binding motif (LKKTET). The expression of TroTβ4 was most abundant in immune organs， and was significantly up-regulated in response to infection bacterial with one of a number of bacteria (including Edwardsiella tarda， Vibrio harveyi， and Streptococcus agalactiae). Purified recombinant TroTβ4 (rTroTβ4) inhibited the growth of bacteria， as measured using an automatic growth curve analyzer， indicating that TroTβ4 has antimicrobial functions. When administered in vivo， overexpression of TroTβ4 in T. ovatus， bacterial colonization of tissues was significantly reduced. In contrast， when a DNA vector-based siRNA technology was used to knock down TroTβ4 expression， bacterial dissemination and colonization of tissues increased significantly. Taken together， these results provide the first in vivo evidence to indicate that teleost thymosin beta 4 plays a significant role in innate antibacterial immune responses in addition to in vitro bacteriostatic activity. This provides valuable information regarding the biological functions of teleost thymosin beta.