Introduction: Myocardial endotoxin tolerance may be induced in both males and females; however, it remains unknown whether there are mechanistic and threshold differences between the sexes. We hypothesized that endogenous estrogen mediates a higher threshold for endotoxin-induced protection in females. Methods: Adult proestrus and ovariectomized (OVX) female rats were preconditioned with intraperitoneal injections of 125 (PC+125) or 500 (PC+500) mcg/kg Salmonella typhimurium lipopolysaccharide (ETX) or normal saline (PC-). 24 hours later injury dose ETX (500 mcg/kg) was injected. After six hours, myocardial function was measured via Langendorff. p38 MAPK and JNK activation as well as TNF, IL-1, and IL-6 expression were evaluated. Results: ETX injury significantly decreased LVDP in PC- groups versus controls. PC+500 protected against ETX injury resulting in normal cardiac function. PC+125 protected OVX but not proestrus females which had diminished myocardial function. Activated JNK and TNF increased in PC- animals, but were diminished in PC+500. Importantly, activated JNK and TNF increased in PC+125 proestrus females, whereas PC+125 OVX females displayed decreases in these molecules. There were no differences in p38 MAPK activation or expression of IL-1 or IL-6. Conclusions: These results demonstrate that proestrus females require a higher stimulus (PC+500) to achieve myocardial protection against ETX injury. Removal of endogenous estrogen (OVX) lowered the preconditioning threshold (PC+125) resulting in protection after lesser injury. Additionally, myocardial JNK and TNF expression was decreased in OVX PC+125 females, which correlated with myocardial function differences. Therefore, we conclude that endogenous estrogen mediates a higher threshold for endotoxin tolerance in female myocardium.