The iron-withholding strategy of innate immunity is an effective antimicrobial defense mechanism that combats microbial infection by depriving microorganisms of Fe3+, which is important for their growth and propagation. Transferrins (Tfs) are a group of iron-binding proteins that exert their antimicrobial function through Fe3+ sequestration. The current study describes both structural and functional characteristics of a transferrin ortholog from rock bream Oplegnathus fasciatus (RbTf). The RbTf cDNA possesses an open reading frame (ORF) of 2079 bp encoding 693 amino acids. It has a molecular mass of approximately 74 kDa and an isoelectric point of 5.4. In silico analysis revealed that RbTf has two conserved domains: N-terminal domain and C-terminal domain. Pairwise homology analysis and phylogenetic analysis revealed that RbTf shared the highest identity (82.6%) with Dicentrarchus labrax Tf. According to the genomic analysis, RbTf possesses 17 exons and 16 introns, similar to the other orthologs. Here, we cloned the N terminal and C terminal domains of RbTf to evaluate their distinct functional features. Results obtained through the CAS (chrome azurol S) assay confirmed the iron-binding ability of the RbTf, and it was further determined that the iron-binding ability of rRbTfN was higher than that of rRbTfC. The antimicrobial functions of the rRbTfN and the rRbTfC were confirmed via the iron-dependent bacterial growth inhibition assay. Tissue distribution profiling revealed a ubiquitous expression with intense expression in the liver. Temporal assessment revealed that RbTf increased after stimulation of LPS, Edwardsiella tarda, and Streptococcus iniae post injection (p.i.). These findings demonstrated that RbTf is an important antimicrobial protein that can combat bacterial pathogens.