Lymphocyte-specific protein tyrosine kinase (Lck) plays a critical role in effective signal transductions that are fundamental to T cell differentiation, proliferation, and effector functions. In this paper, the Lck gene of Nile tilapia, Oreochromis niloticus (designated as On-Lck), was cloned and its expression pattern under the stimulation of Streptococcus agalactiae was investigated. Sequence analysis showed important structural characteristics required for T cell receptor (TCR) signal transduction were detected in the deduced amino acid sequence of On-Lck, and the deduced genomic structure of On-Lck was similar to the known Lck. In healthy Nile tilapia, the On-Lck transcripts were mainly detected in the thymus, spleen, head kidney, and gill. When immunized with inactivated S. agalactiae, the On-Lck mRNA expression was significantly upregulated in the thymus, spleen, and head kidney. Moreover, there was a clear time-dependent expression pattern of On-Lck after immunization, and the expression reached the highest level at 48 h in the spleen and thymus and at 72 h in the head kidney, respectively. This is the first report on the expression of Lck induced by intracellular bacteria vaccination in teleosts. These findings indicated that On-Lck may play an important role in the immune response to intracellular bacteria in Nile tilapia.