This study compared the effects of di(n‐butyl) phthalate (DBP) on the oxidative damage and antioxidant enzymes activity in testes of hyperthyroid rats. Hyperthyroidism was induced in pubertal male rats by intraperitoneal injection of triiodothyronine (T3, 10 μg/kg body weight) for 30 days. An oral dose of DBP (750 mg/kg) was administered simultaneously to normal or hyperthyroid (T3) rats over a 30‐day period. No changes in body weight were observed in the hyperthyroid groups (T3, T3 + DBP) compared with controls. There were significantly higher serum T3 levels observed in the hyperthyroid rats than in the control, but the serum thyroid stimulating hormone levels were markedly lower in the hyperthyroid rats. DBP significantly decreased the weight of the testes in the normal (DBP) and hyperthyroid (T3 + DBP) groups. The serum testosterone concentrations were significantly lower in only DBP group. DBP significantly increased the 8‐hydroxy‐2‐deoxyguanosine (8‐OHdG) level in the testes, whereas the DBP‐induced 8‐OHdG levels were slightly higher in T3 + DBP group. Superoxide dismutase and glutathione peroxidase activities were significantly higher in the testes of the DBP or T3 + DBP groups. Catalase (CAT) activity was significantly higher in the DBP treatment group, but the T3 + DBP group showed slightly lower DBP‐induced CAT activity. The testicular expression of thyroid hormone receptor α‐1 (TRα‐1) was significantly higher in the DBP groups, and androgen receptor (AR) expression was not detected in the DBP treatment group. In addition, DBP significantly increased the peroxisome proliferator‐activated receptor‐r (PPAR‐r) levels in the testis. These results suggest that hyperthyroidism can cause a change in the expression level of PPAR‐r in testes, and may increase the levels of oxidative damage induced by the metabolic activation of DBP. © 2007 Wiley Periodicals, Inc. Environ Toxicol 22: 245–255, 2007.