The function of activating transcription factor 1 (ATF1) and the mechanism about why ATF1 was over-phosphorylated in nasopharyngeal carcinoma (NPC) progression is completely undiscovered. In this study, a series of experiments both in vitro and in vivo were used to characterize a promotive function of ATF1 in NPC tumorigenesis and identify prolyl isomerase Pin1 as a novel regulator of ATF1 at post-transcription. First, we found that overexpression of ATF1 promoted colony formation in NPC. However, the high protein level of ATF1 in NPC was not resulted from high mRNA level. Then, a direct interaction between Pin1 and ATF1 at Thr184 was demonstrated using mammalian two-hybrid assay and coimmunoprecipitation. Cycloheximide (CHX) treatment indicated Pin1 stabilized the expression of ATF1 at post-transcription level. We confirmed that Pin1 upregulated ATF1 transcriptional activity of Bcl-2 using luciferase reporter assay, quantitative RT-PCR and western blot. Furthermore, the newly identified phosphorylation of ATF1 at Thr184 was suggested to have an important role in ATF1 function of transcription and tumor promotion. Finally, high expression of Pin1 in NPC tissue was found to be positively correlated with ATF1. The ATF1 promoted NPC tumorigenesis was regulated by Pin1 both in vitro and in vivo. All these findings clearly state that Pin1 is a novel regulator of ATF1 at Thr184 and thereby enhances ATF1 transcription activity and tumorigenesis promotive function in NPC.