Nonylphenol (NP) is one widely distributed representative of environmental estrogens that disturb reproductive activities, bone metabolism and brain function through interfering diverse signal pathways leading to hormone metabolic dysfunctions, immunologic derangement, and tumorigenesis. Few of previous studies have observed the subacute toxicity on rodents, and little has been focused on the mechanism underneath the toxicities observed.The 32 male Sprague-Dawley (SD) rats were randomly divided into four groups, the negative control group (corn oil) NP low, medium and high dose groups [30, 90, 270 mg/(kg·d)]. SD rats administrated with different dosage of NP every other day for 28d. Elisa and RT-PCR was employed to observe estrogen metabolism markers or mRNA expressions.In serum, NP exposure caused testosterone (T) (p < 0.001), progesterone (PROG) (p < 0.05) and estrone (E1) (p < 0.05) increased. In testicle, NP exposure caused T (p < 0.001), PROG (p < 0.05), E1 (p < 0.05), 17β-estradiol (E2) (p < 0.05) and ERα mRNA (p < 0.01) increased, while P450 aromatizing enzyme (p < 0.001) decreased in NPL and ERβ mRNA (p < 0.001) decreased in NPM and NPH. In liver, NP exposure caused 17β-HSD2 mRNA (p < 0.01) increased, while P450 aromatizing enzyme decreased (p < 0.05).NP exposure exhibited general and estrogenic toxicity in rats through disturbing estrogen secretion network and estrogen receptor expression network, inducing abnormal metabolism of estrogen, whether in serum, liver and testicle.