Monocytes are antigen-presenting cells (APCs) in mammals. Antigen presentation by monocytes via costimulatory molecules was recently confirmed in the Japanese pufferfish Fugu rubripes (also known as Takifugu rubripes) (Sugamata et al., 2009[1]). However, no detailed investigations examining how cytokine gene expression regulates the activation/differentiation of these cells have been conducted to date. Therefore, in this study, the expression of cytokine genes in Fugu monocytes stimulated with TLR agonists (LPS, polyI:C, and IMQ) was profiled. First, the morphological changes and phagocytic activity of stimulated monocytes were examined. At 5 days post-stimulation, the ratio of activated to inactivated monocytes increased (as determined by FCM analysis), and the phagocytic activity of the cells was higher (5-15%) than that of nonactivated cells. Analysis of cytokine gene expression in stimulated monocytes revealed that the genes encoding CSF-1b and IFN-γ are important cytokines in activation/differentiation of monocytes/macrophages, and that genes encoding inflammatory cytokines such as IL-1β, IL-6, IL-18, and TNF-α are upregulated by stimulation. The present study revealed the types of cytokines expressed in Fugu monocytes stimulated with TLR agonists.