Antigen-specific stimulation of T helper (Th) cells initiates signaling cascades that ultimately result in the activation of the transcription factors NF-κB, NFAT, and AP-1 which regulate, together with other factors, many T-cell functions such as cytokine production, proliferation, and differentiation. Ordered assembly and different phosphorylation events, along with subcellular translocation of the CARMA1/Bcl-10/MALT1 complex, determine NF-κB activation after T-cell receptor (TCR) triggering. We now provide evidence that inhibition of the Ser/Thr phosphatase calcineurin (CaN) prevents dephosphorylation of Bcl-10. CaN, in constant interaction with the Bcl-10/MALT1 complex, is able to dephosphorylate Bcl-10. The CaN inhibitor cyclosporine A (CsA) converts a transient phosphorylation of Bcl-10 Ser138 during the immediate early phase of T-cell activation into a persistent state. Thus, subsequent processes such as IKKβ phosphorylation, IκBα degradation, p65 nuclear translocation, and DNA binding are diminished. Consistently, CsA treatment does not affect the phosphorylation pattern of the upstream kinase PKCθ. Together, our findings demonstrate that CaN functions as a critical signaling molecule during Th cell activation, regulating Bcl-10 phosphorylation and thereby NF-κB activation.