Aims: The pleckstrin homology domain leucine-rich repeat protein phosphatase 1 (PHLPP1) specifically regulates phospho-Ser473 of protein kinase B (PKB, Akt) opposing cell survival during myocardial ischemia/reperfusion (I/R). Previous studies demonstrated PHLPP1 expression level was controlled by several mechanisms. However, the regulation mechanism of cardiac PHLPP1 expression following myocardial I/R remains unknown. Main methods: The current study utilized the mouse model of myocardial I/R injury in vivo and the neonatal rat ventricular myocytes (NRVMs) of hypoxia/reoxygenation (H/R) injury in vitro. Expression of PHLPP1, nuclear factor-kappa B (NF-kappa B) and pNF-kappa B were determined by western blot. The expression of PHLPP1 and translocation of NF-kappa B was assessed by immunofluorescence. Chromatin immunoprecipitation (ChIP) assay was used to detect the binding of NF-kappa B to the promoter region of phlpp1 gene. Key findings: Myocardial I/R had no effect on cardiac PHLPP1 expression following I/R (30 min/2 h) but decreased after 4 h reperfusion. In vitro, H/R (4 h/1 h) and tumor necrosis factor-alpha (TNF-alpha)-stimulation resulted in upregulation of PHLPP1 in NRVMs, which was blocked with etanercept. Yet, H2O2-induced oxidative stress had no obvious effect on PHLPP1 expression of NRVMs at early stage but N-acetylcysteine (NAC) pretreatment increased PHLPP1 levels after 4 h H2O2 stimulation. TNF-a and H/R led to both expression and transcriptional activity of NF-kappa B,accompany with higher expression of PHLPP1. Pyrrolidine dithiocarbamate (PDTC), a NF-kappa B inhibitor, prevented the response not only in TNF-alpha-treated cardiomyocytes but also in H/Rtreated group. Significance: These results implicated that TNF-alpha involved in cardiac PHLPP1 upregulation during reoxygenation, which was mediated by NF-kappa B transcriptional activity.