Ophiopogon japonicus polysaccharide (FOJ-5) from Radix ophiopogonis has shown anti-myocardial ischemic action in vitro and in vivo in our previous studies. In order to clarify the influence of chemical modifications on the action, a series of sulfated FOJ-5 (FOJ-5-S) with different substitution degrees were prepared and the anti-myocardial ischemic action of the natural FOJ-5 and the FOJ-5-S were studied in vitro and in vivo. Langendorff isolated rat hearts and acute myocardial ischemic rats induced by isoprenaline were employed as myocardial ischemic models in our experiments. The amplitude and frequency of cardiac contraction, coronary blood flow at different time points after ischemia/reperfusion were measured in vitro. The ST segment shift in electrocardiogram and lactate dehydrogenase level in blood plasma were observed on the in vivo model. The results indicated that FOJ-5 and FOJ-5-S had the anti-myocardial ischemic action compared with non-treated vehicle groups. Furthermore, it was found that FOJ-5-S had significant action on the in vivo model compared with FOJ-5 (P < 0.05). And the obtained results from the further study also indicated that only when the degree of substitution was in a certain range, the FOJ-5-S had excellent anti-myocardial ischemic activity.