Toll-like receptors (TLRs) function as pattern-reorganization receptors in mammals and play an essential role in innate immunity. After recognizing certain ligands, TLRs activate a cascade of signal pathways to establish a guard environment. For the first time, we report that in vitro treatment with recombinant calcineurin subunit B (CNB) upregulated several TLR-related genes. Calcineurin subunit B interacted with the ectodomain of TLR4 in vitro. On further investigation, phosphorylation of interferon regulatory factor 3 and degradation of IκB-α were observed by CNB stimulation. In addition to pro-inflammatory cytokines, transcription and production of β-interferon were also enhanced after CNB stimulation. Thus, CNB could be further explored as a cancer and virus immunotherapy drug. (Cancer Sci 2012; 103: 515–521)