The transcription factor NF-κB is activated by the degradation of its inhibitor IκBα, resulting in its nuclear translocation. However, the mechanism by which nuclear NF-κB is subsequently regulated is not clear. Here we demonstrate that NF-κB function is regulated by Pin1-mediated prolyl isomerization and ubiquitin-mediated proteolysis of its p65/RelA subunit. Upon cytokine treatment, Pin1 binds to the pThr254-Pro motif in p65 and inhibits p65 binding to IκBα, resulting in increased nuclear accumulation and protein stability of p65 and enhanced NF-κB activity. Significantly, Pin1-deficient mice and cells are refractory to NF-κB activation by cytokine signals. Moreover, the stability of p65 is controlled by ubiquitin-mediated proteolysis, facilitated by a cytokine signal inhibitor, SOCS-1, acting as a ubiquitin ligase. These findings uncover two important mechanisms of regulating NF-κB signaling and offer new insight into the pathogenesis and treatment of some human diseases such as cancers.