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Dry tuberous roots of ophiopogon japonicus are famous Chinese herbal medicines. Several studies have been shown that polysaccharide is one of the main active ingredients of ophiopogon japonicus and plays an important role in pharmacological action. In the study, the effects of polysaccharides from ophiopogon japonicus (POJ) on the oxidative damage induced by strenuous exercise were investigated. Male mice were randomly divided into four groups and designated as high-dose POJ-treated, medium-dose POJ-treated, low-dose POJ-treated and control groups, which received 80, 40, and 20 mg/kg POJ and physiological saline, respectively, by gavage once a day for 28 days. This was followed by an exhaustive running exercise and measurements of various biochemical parameters from blood and muscles tissue. The results showed that POJ could significantly prolong the exhaustive running time of mice, decrease the levels of lactate dehydrogenase (LDH), creatine kinase (CK) and 8-Hydroxy-2′-deoxyguanosine (8-OHdG) in blood, and malondialdehyde (MDA) levels in muscle, which were accompanied by corresponding increased levels of superoxide disproportionation enzyme (SOD), glutathione peroxidase (GPx), and catalase (CAT) in muscle. The data suggest that POJ have protective effects on strenuous exercise-induced oxidative damage.