Abstract: Perfluorododecanoic acid (PFDoA), a ubiquitous contaminant detected in environmental matrices, wildlife, and human blood, has been shown to produce adverse effects on male reproduction in rats. The mechanism of action of PFDoA in testis, however, is not well understood. In the present study, male rats were orally exposed to PFDoA (0.02, 0.2, and 0.5mg/kg/day for 110 days), and a two-dimensional gel electrophoresis (2-DE) approach was employed to investigate the alteration of protein expression in the testes. Decreased serum progesterone levels were observed. Matrix-assisted laser desorption/ionization (MALDI) tandem time of flight (TOF/TOF) mass spectrometry analysis allowed the unambiguous identification of 40 differentially expressed proteins. These proteins are mainly involved in mitochondrial respiration, oxidative stress, sperm activity, cytoskeleton and intracellular signal transduction. Furthermore, PFDoA led to decreases in activities of superoxide dismutase (SOD), mitochondrial H-ATPase, and cytochrome c oxidase as well as to an increase in lipid peroxidation in testes. Our results indicated that these proteins, which are involved in mitochondrial respiratory and antioxidative responses, play important roles in the inhibition of testicular steriodogenesis in response to PFDoA. Our data demonstrate that alterations of multiple pathways may be associated with the toxic effects of PFDoA on testes. SOD and H-ATPase subunit d may be sensitive to PFDoA exposure in testis.