This study aimed to investigate the effects of anti-tumor necrosis factor (TNF)-α antibody (Ab) on alteration of penile structure in the hyperprolactinemia (hyperPRL) rat model. HyperPRL was induced in 8-week-old male Sprague-Dawley rats by allografting anterior pituitary (AP) glands under the renal capsule (+AP rats). Rats implanted with cerebral cortex (CX) were used as sham control (+CX rats). At 6 weeks post implantation, rats received either a single intra-testicular dose of TNF-α Ab (12.5 μg/kg) or testosterone replacement (2 doses of testosterone enanthate [TE], 3 mg/kg), and they were sacrificed 1 week later. Blood and penile tissue was collected for analysis. Compared to +CX rats, the +AP group had lower serum testosterone concentration and neuronal nitric oxide synthase (nNOS) expression, but exhibited a higher ratio of collagen III/I in the corpus cavernosum. Smooth muscle content exhibited no significant changes. At 1 week post TNF-α Ab injection, the collagen III/I ratio in the +AP group was decreased, and the smooth muscle content and nNOS expression increased significantly. These findings were comparable to those observed in +AP rats receiving TE. Testicular TNF-α suppresses testosterone release, which in turn results in the erectile dysfunction (ED) seen in hyperPRL. Intra-testicular TNF-α Ab treatment is as effective as testosterone supplementation on penile structure normalization in the hyperPRL model.