We have previously identified a stop-gain mutation in PPP3CB, which encodes a catalytic subunit of calcineurin, in a primary neuroblastoma (NB) with MYCN amplification by whole-exome sequencing of 57 paired neuroblastoma samples. The stop-gain mutation identified in PPP3CB results in a constitutive active form of the PPP3CB protein. In this study, we investigated the clinical and functional roles of PPP3CB in NB. Expression levels of PPP3CB in 72 NB clinical samples were measured by quantitative RT-PCR method. We found that high expression of PPP3CB was significantly associated with poor prognosis in our sample set. Multivariate Cox regression analysis showed that PPP3CB was an independent prognostic factor predicting poor outcome. On the other hand, overexpression of wild-type and mutated PPP3CB promoted cell growth, while PPP3CB knockdown decreased cell growth in NB cells. Enforced expression of mutated PPP3CB enhanced anchorage-independent growth in NB cells. Both wild-type and mutated PPP3CB activated the Nuclear Factor of Activated T cell (NFAT) signaling, accompanied with activation of Akt and inhibition of the GSK3β activity, which were inhibited by treatment with calcineurin inhibitors. Treatment with calcineurin inhibitors suppressed cell proliferation and induced apoptotic cell death in several NB cell lines. Thus, our data indicate that as a catalytic subunit of calcineurin, PPP3CB is an independent indicator of NB predicting poor prognosis, which may contribute to malignant biological behaviors of NB cells through activation of NFAT signaling. Inhibition of calcineurin activity might have therapeutic potential against high-risk NB.