Interleukin 6 (IL-6), a pleiotropic cytokine, plays an important role in humoral immune response, not only inducing the differentiation of B cells into plasma cells, but also promoting antibody-secreting cells (ASCs) to produce antibodies. In this study, Nile tilapia (Oreochromis niloticus) IL-6 (OnIL-6) was identified and characterized at expression level in response to bacterial infection and promotion of antibody production. The open reading frame of OnIL-6 ORF is consisted of 663 bp encoding a polypeptide of 220 amino acids. The deduced OnIL-6 protein contained an IL-6/G-CSF family signature, two conserved cysteine, and four α-helix bundles, which was highly homologous to other species. Spatial mRNA expression analysis revealed that the highest expression of OnIL-6 was observed in the thymus. After in vivo challenges of lipopolysaccharide (LPS) and Streptococcus agalactia (S. agalactiae), OnIL-6 expressions were significantly up-regulated in head kidney and spleen. The similar up-regulation of OnIL-6 was observed in the head kidney and spleen leukocytes in vitro stimulation with LPS and S. agalactiae. In addition, inducement with the recombinant OnIL-6 ((r)OnIL-6) in vitro caused significant increases in expressions of both sIgM and mIgM. Moreover, the (r)OnIL-6 stimulation enhanced the secretion of sIgM (more especially in P50 plasma-like B cells) and the production of mIgM in P60 and P70 B cell subsets (resting B cells, activated B cells and plasmablast-like B cells) in vitro. Taken together, this study indicated that OnIL-6 might be involved in host defense against bacterial infection and promote the production of antibody in Nile tilapia.