As a pleiotropic cytokine in the interleukin (IL)-12 family, IL-27beta plays a significant role in regulating immune cell responses, eliminating invading pathogens, and maintaining immune homeostasis. Although non-mammalian IL-27beta homologs have been identified, the mechanism of whether and how it is involved in adaptive immunity in early vertebrates remains unclear. In this study, we identified an evolutionarily conserved IL-27beta (defined as OnIL-27beta) from Nile tilapia (Oreochromis niloticus), and explored its conserved status through gene collinearity, gene structure, functional domain, tertiary structure, multiple sequence alignment, and phylogeny analysis. IL-27beta was widely expressed in the immune-related tissues/organ of tilapia. The expression of OnIL-27beta in spleen lymphocytes increased significantly at the adaptive immune phase after Edwardsiella piscicida infection. OnIL-27beta can bind to precursor cells, T cells, and other lymphocytes to varying degrees. Additionally, IL-27beta may be involved in lymphocyte-mediated immune responses through activation of Erk and JNK pathways. More importantly, we found that IL-27beta enhanced the mRNA expression of the Th1 cell-associated cytokine IFN-gamma and the transcription factor T-bet. This potential enhancement of the Th1 response may be attributed to the activation of the JAK1/STAT1/T-bet axis by IL-27beta, as it induced increased transcript levels of JAK1, STAT1 but not TYK2 and STAT4. This study provides a new perspective for understanding the origin, evolution and function of the adaptive immune system in teleost.