Objective: Hydrogen peroxide (H2O2) is a potent reactive oxygen species that causes cardiomyocytes injury.As an important cytokine,interleukin 6 (IL-6) has cardioprotective effects as it plays an essential role in the late phase of preconditioning.Our work is to investigate if IL-6 preconditioning has protective effects on neonatal rat ventricular cardiomyocytes in response to H2O2 and its underlying mechanism.Methods: Gel-based comparative proteomic approach along with small interfering RNA (siRNA) and Western blot analysis was used to analyse mechanisms of IL-6 preconditioning on H2O2-induced neonatal rat ventricular cardiomyocytes injury.Results: IL-6 preconditioning protected eardiomyocytes against H2O2-induced cell death.Proteomic analysis showed that IL-6 pretreatment further increased the expression of prohibitin and improved the viability of cardiomyocytes exposed to H2O2.Knocking down of prohibitin with siRNA abrogated this protection by increasing apoptosis rate.Tyrosine kinase inhibitor AG490 decreased signal transducers and activators of transcription 3 (STAT3) phosphorylation and down-regulated prohibitin expression in cardiomyocytes pretreated with IL-6 and exposed to H2O2, which further dampened the protective effects of IL-6 preconditioning.Conclusion: Our results provide direct evidence that prohibitin is a protective factor of IL-6 preconditioning in H2O2-induced neonatal rat ventricular cardiomyocytes death.The upregulation of prohibitin by IL-6 is, at least, partially regulated through STAT3 phosphorylation.Copyright (C) 2012 John Wiley & Sons, Ltd.