Pharmacological studies have shown that signal transducers and activators of transcription (STATs) are necessary for the delayed cardioprotection of ischemic preconditioning (PC). However, pharmacologic STAT inhibitors are not specific; furthermore, the individual role of STAT3 in late PC remains unknown. The objectives of the study were (i) to create an inducible, cardiac-specific STAT3 knockout mouse; (ii) to verify whether STAT3 deletion has any adverse effects in the short term (~1month); and (iii) to use this novel tool to evaluate the role of STAT3 in the PC-induced upregulation of cardioprotective and anti-apoptotic proteins. We created an inducible, cardiomyocyte-restricted STAT3 deficient mouse (MCM TG:STAT3flox/flox) by interbreeding STAT3flox/flox mice and tamoxifen-inducible MCM TG mice. Treatment of MCM TG:STAT3flox/flox mice with tamoxifen resulted in deletion of STAT3 specifically in cardiac myocytes, concomitant with abrogation of ischemic PC-induced Tyr-705 and Ser-727 phosphorylation of STAT3 and increased STAT3 DNA-binding activity. In vehicle-treated MCM TG:STAT3flox/flox mice, ischemic PC increased the expression of cardioprotective (COX-2 and HO-1) and anti-apoptotic (e.g., Mcl-1, Bcl-xL, c-FLIPL, c-FLIPS) proteins 24h later; in contrast, in tamoxifen-treated MCM TG:STAT3flox/flox mice this increase was completely absent. Deletion of STAT3 had no apparent adverse effects on LV structure or function after 35days. We have developed a novel inducible, cardiomyocyte-restricted STAT3 deficient mouse that can be used to specifically interrogate the role of this transcription factor in cardiovascular pathophysiology in vivo. Our data demonstrate, for the first time, that recruitment of STAT3 plays an obligatory role in the upregulation of cardioprotective and anti-apoptotic proteins and suggest that STAT3 activation is important in inhibiting both the death receptor pathway (which is modulated by c-FLIPL and c-FLIPS) and the mitochondrial pathway (which is mediated by Mcl-1 and Bcl-xL).