The aim of the present study was to evaluate the protective effects of isoflavone (ISO) against zearalenone (ZEA) residues in the muscle and liver tissues of prepubertal gilts. Seventy 75-day-old, prepubertal, female pigs (Duroc × Landrace × Yorkshire, 26.5 ± 0.60 kg) were allocated randomly to seven diet treatments for 21days as follows: one control group (fed the basal diet) and six groups fed the basal diet with the addition of either 0.5 or 2.0 mg/kg ZEA plus either 0, 300 or 600 mg/kg ISO. The results showed that the diet with 2.0 mg/kg ZEA added caused an increase of ZEA residue level in muscle tissue (P < 0.05), and that the addition of both 0.5 and 2.0 mg/kg ZEA increased the residue level of ZEA in the liver of prepubertal gilts (P < 0.05). Addition of 600 mg/kg ISO to 2.0 mg/kg ZEA-contaminated diet decreased the ZEA residue level in liver tissue (P < 0.05), and the addition of 300 or 600 mg/kg ISO to the 2.0 mg/kg ZEA-contaminated diet decreased the residue levels of ZEA in muscle tissue (P < 0.05). Western blot analysis demonstrated that feeding ZEA to prepubertal gilts increased their protein expression of 3α/3β-hydroxysteroid dehydrogenase (HSD; P < 0.05), and that the addition of 300 or 600 mg/kg ISO to the 2.0 mg/kg ZEA-contaminated diet decreased the protein expression of 3α/3β-HSD (P < 0.05), compared with the addition of 2.0 mg/kg ZEA alone. The results demonstrated that muscle and liver tissues retain residual ZEA when pigs are fed a diet contaminated with high concentrations of ZEA, and that the concentration of ZEA in muscle and liver tissues increased with increased amounts of ZEA in the feed. In diets contaminated with high levels of ZEA, the addition of ISO may accelerate the biotransformation and degradation of ZEA and its metabolites, and reduce the residues of ZEA in liver and muscle tissues of prepubertal gilts.