To determine the detrimental role of tetrachlorodibenzo-p-dioxin (TCDD) in testicular histology, spermatogenesis-related panels and proteome, and serum sex hormone levels. In all, 40 male rats were divided into equal groups: a normal control (NC) group that received vehicle and saline, and a TCDD-treated (TT) group injected intraperitoneally with TCDD (one dose, 50 mu g/kg body weight). The rats were killed 4 weeks after TCDD exposure and testicular weight, histopathology, proteome and variables related to spermatogenesis, and serum sex hormone levels were investigated. TCDD induced a significant decrease in testicular weight, Johnsen's score, seminiferous tubular size, percentage of tubules containing sperm, sperm counts, germ cell counts and Sertoli cell index. In addition, there was a significant decrease in serum testosterone level (P < 0.01) and a remarkable increase in oestradiol (P < 0.01), follicle-stimulating hormone (P < 0.05) and luteinizing hormone (P < 0.05) levels in the TT group. The expression of six testicular proteins including testis-specific heat shock protein (Hsp70), protein disulphide isomerase A3 precursor, 3-phosphoglycerate dehydrogenase, nonmuscle myosin heavy-chain type B-like protein, and superoxide dismutase 1 were significantly up-regulated (P < 0.05-0.01). Interestingly, fertility protein SP22 and phosphatidylethanolamine-binding protein were down-regulated but this was only significant for fertility protein SP22 (P < 0.05). TCDD induces marked histological changes in the testis, impairs variables related to spermatogenesis, and increases serum oestradiol levels but decreases testosterone levels. In particular, TCDD disturbs testicular proteome profiles in rats.