The peptidyl-prolyl isomerase Pin1 is frequently up-regulated in human cancers in which Rel/nuclear factor-κB (NF-κB) is constitutively activated, but its role in these cancers remains to be determined, and evidence is still lacking to show that Pin1 contributes to cell transformation by Rel/NF-κB. Rel/NF-κB transcriptional and oncogenic activities are modulated by several posttranslational modifications and coregulatory proteins, and previous studies showed that cytokine treatment induces binding of Pin1 to the RelA subunit of NF-κB, thereby enhancing RelA nuclear localization and stability. Here we show that Pin1 associates with the Rel subunits of NF-κB that are implicated in leukemia/lymphomagenesis and modulates their transcriptional and oncogenic activities. Pin1 markedly enhanced transformation of primary lymphocytes by the human c-Rel protein and also increased cell transformation by the potent viral Rel/NF-κB oncoprotein v-Rel, in contrast to a Pin1 mutant in the WW domain involved in interaction with NF-κB. Pin1 promoted nuclear accumulation of Rel proteins in the absence of activating stimuli. Importantly, inhibition of Pin1 function with the pharmacologic inhibitor juglone or with Pin1-specific shRNA led to cytoplasmic relocalization of endogenous c-Rel in human lymphoma-derived cell lines, markedly interfered with lymphoma cell proliferation, and suppressed endogenous Rel/NF-κB–dependent gene expression. Together, these results show that Pin1 is an important regulator of Rel/NF-κB transforming activity and suggest that Pin1 may be a potential therapeutic target in Rel/NF-κB–dependent leukemia/lymphomas. [Cancer Res 2009;69(11):4589–97]