The innate immune system is the first line of defense against infection by pathogens. It consists of various elements, including Toll-like receptors (TLRs), which recognize molecular patterns associated with pathogens and trigger the immune response, through activation of important transcription factors such as NF-κB, which are usually found sequestered in the cytoplasm by IκBα until it receives the release signal. Piscirickettsia salmonis causes piscirickettsiosis or salmonid rickettsial septicemia, a disease of great importance in Chile, representing 79.4% of the secondary mortality in important species such as Salmo salar, which is reflected in the Chilean economy. Prolactin (PRL) is a peptide hormone which has immunomodulating functions in mammals and some fish. Olavarría et al. (2010, J Immunol 185:3873-3883) determined its ability to increase the respiratory burst, its relationship with the JAK/STAT pathway, and the expression of interleukin IL-1β in Sparus aurata. Therefore, the present study was intended to establish a possible correlation and modulation between the signal transduction pathway of PRL (JAK/STAT), the pathways of NF-κB, and TLRs, in an infection caused by P. salmonis in salmon head kidney (SHK‑1) cells of S. salar. Stimulus with native PRL from S. salar was performed, and gene expression was analyzed for IL-1β, IκBα, TLR1, and TLR5M (membrane-bound form). In addition, the effect of PRL in the nuclear translocation of the transcription factor NF-κB and the possible involvement of JAK2 were analyzed by using a pharmacological inhibitor of this kinase. The results show a positive modulation of PRL in all analyzed genes and a significant increase in the translocation of NF-κB, recording a maximum at 2 h post-treatment, supporting the stimulatory hypothesis of PRL.