Selenoprotein P (SeP), the major selenoprotein in plasma, is produced mainly by the liver, although SeP expression is detected in many organs. Recently, we reported stimulation of SeP promoter activity by the forkhead box transcription factor FoxO1a in hepatoma cells and its attenuation by insulin. Here, we demonstrate that this translates into fine‐tuning of SeP production and secretion by insulin. Overexpression of peroxisomal proliferator activated receptor‐γ coactivator 1α (PGC‐1α) enhanced the stimulatory effect of FoxO1a on SeP promoter activity. We identified a novel functional binding site for hepatocyte nuclear factor (HNF)‐4α, termed hepatocyte nuclear factor binding element 1, in the human SeP promoter directly upstream of the FoxO‐responsive element daf16‐binding element 2 (DBE2). Point mutations in hepatocyte nuclear factor binding element 1 alone or together with DBE2 decreased basal activity and responsiveness of the SeP promoter to PGC‐1α. Moreover, the PGC‐1α‐inducing glucocorticoid dexamethasone strongly enhanced SeP messenger RNA levels and protein secretion in cultured rat hepatocytes, whereas insulin suppressed the stimulation of both PGC‐1α and SeP caused by dexamethasone treatment. In a brain‐derived neuroblastoma cell line with low basal SeP expression, SeP transcription was stimulated by PGC‐1α together with FoxO1a, and overexpression of HNF‐4α potentiated this effect. Conclusion: High‐level expression of SeP in liver is ensured by concerted action of the coactivator PGC‐1α and the transcription factors FoxO1a and HNF‐4α. Hence, the production of SeP is regulated similarly to that of the gluconeogenic enzyme glucose‐6‐phosphatase. As hepatic SeP production is crucial for selenium distribution throughout the body, the present study establishes PGC‐1α as a key regulator of selenium homeostasis. (HEPATOLOGY 2008;48:1998‐2006.)