The nucleotide-binding domain leucine-rich repeat proteins (NLRs), a class of innate immune receptors that respond to pathogen attack or cellular stress, have gained increasing attention. NLRC5 is the largest member of NLR family, which has recently been identified as a critical regulator of immune responses. In this study, we explore the role of NLRC5 in cytokine secretion and the role of the JAK2/STAT3 signaling pathway in lipopolysaccharide-induced NLRC5 expression in RAW264.7 cells. We demonstrated that overexpression of NLRC5 results in a downregulation of interleukin-6 (IL-6) and tumor necrosis factor-α (TNF-α) secretion; on the other hand, knockdown of NLRC5 by transfecting siRNA enhanced IL-6 and TNF-α secretion in RAW264.7 cells. These results indicated that NLRC5 plays a negative role in the regulation of IL-6 and TNF-α. Meanwhile, AG490 (a specific inhibitor of the JAK2/STAT3 signaling pathway) and JAK2 siRNA were used to manipulate JAK2/STAT3 activity. Finally, the results showed that AG490 and JAK2 siRNA inhibited NLRC5 expression and the expression levels of p-JAK2 and p-STAT3. We, for the first time, demonstrate that the inhibition of the JAK2/STAT3 signaling pathway results in decrease of NLRC5 expression.