Pharmacological postconditioning offers a clinical perspective for all patients with ischemic heart disease. Penehyclidine hydrochloride (PHC) is a new type of anticholinergic drug. We previously reported that PHC preconditioning protects against I/R injury in rat hearts in vivo. Ischemic heart disease often occurs suddenly, so postconditioning is more significant than preconditioning. However, studies evaluating myocardial protective effects of PHC postconditioning are unavailable. We explored the effects and time-window of cardioprotection of PHC postconditioning in myocardial I/R injury. PHC was administered by intravenous at various times (t = −5, 0, 5, 10, 15, or 30 min) after the onset of reperfusion in addition to I/R rat. We observed five different indicators including infarct size, inflammatory response, myocardial enzyme, oxidative stress, and Ca2+ overload to quantify the effect of cardioprotection. Evans blue and TTC staining were used to measure myocardial infarct size. The expression of NF-κ B and IκB-α was analyzed using Western blot. ELISA was conducted to detect inflammatory and anti-inflammatory mediators. The Ca2+ level was determined using assay kit. PHC postconditioning (from −5 to 10 min after the onset of reperfusion) significantly reduced infarct size, downregulated NF-κ B expression, and decreased the release of inflammatory mediators, while significantly upregulating IκB-α expression and increasing the release of anti-inflammatory mediators. All PHC postconditioning groups significantly reduced Ca2+ level. PHC postconditioning is cardioprotective over a larger time-window (from −5 to 10 min after the onset of reperfusion). The probable mechanism is inhibition of NF-kB regulated inflammatory response pathway.