Safe and potent adjuvants are required to establish effective vaccines. In the present work, we show that calcineurin subunit B promotes the secretion of pro-inflammatory cytokines, such as tumor necrosis factor-α, interleukin-12p70 (IL-12 p70), IL-6 and the chemokine IL-8. It also up-regulates transcript levels of chemokines in bone marrow-derived dendritic cells. In an animal model, C57BL/6 mice were divided into four groups, immunized with ovalbumin (OVA), OVA mixed with calcineurin subunit B (CnB), CnB and PBS, respectively. The splenocytes from mice immunized with OVA in combination with CnB produced higher levels of IFN-γ and CTL when in vitro stimulated with OVA protein. Subcutaneous (s.c.) immunization of C57BL/6 mice with OVA plus CnB conferred greater protection against tumor-forming E.G7-OVA cells than did injection of OVA alone, and the survival rate of mice immunized intraperitoneally was higher than that of mice immunized s.c. Thus, CnB exerts potent adjuvant effects that polarize responses toward T(h)1 and elicit anti-tumor and anti-infection immunity. CnB could be of use as a prophylactic adjuvant as it is a human-derived safe agent and has immune-modulating effects that promote the control of cancer and infectious diseases.