Post-translational modification of peptidyl cis/trans prolyl isomerase Pin1 is crucial in regulation of gene stability. Pin1 phosphorylation at Ser(16) has been regarded as a marker for Pin1 isomerase activity and introduction of phosphorylation on Ser/Thr-Pro of substrate proteins is prerequisite for its binding activity with Pin1 and subsequent isomerization. Here, we found that 90 kDa ribosomal protein S6 kinase 2 (RSK2) could form a physical complex with Pin1, leading to phosphorylation of Pin1 at Ser(16) ex vivo and in vitro respectively. Intriguingly, Pin1(+/+) mouse embryonic fibroblasts (MEFs) exhibited significantly an increase in 12-O-tetradecanoylphorbol-13-acetate (TPA)-induced RSK2 phosphorylation with a marginal Pin1 phosphorylation compared with Pin1(-/-) MEFs. Moreover, TPA-induced Ser(16) Pin1 phosphorylation as well as RSK2 phosphorylation was considerably profound in RSK(+/+) MEFs but not in RSK(-/-) MEFs. Consequently, knockdown of Pin1 using shRNA-Pin1 suppressed TPA-induced cell transformation in JB6 CI41 cells. Overall, these results indicate that Pin1 plays a critical role in TPA-induced tumorigenesis plausibly via physical interaction with RSK2 and reciprocal phosphorylation, therefore suggesting a potential therapeutic target for cancer treatment.