There is a growing body of evidence to support the direct link between obstructive bladder dysfunction and erectile dysfunction (ED). However, there have been few pathophysiological studies to determine the relationship between lower urinary tract syndrome (LUTS) and ED. As the transforming growth factor-β1 (TGF-β1) that induces the synthesis of collagen in the penile tissues is critical for the development of ED, the first aim of this study was to investigate the expression of TGF-β1 in the penis from male rabbits with chronic partial bladder outlet obstruction (PBOO). Besides, it has been suggested that oxidative stress plays a significant role in the pathophysiological mechanism of ED. Thus, the second aim of this study was to further investigate whether the urinary or serum oxidative stress markers are involved in chronic PBOO-induced penile dysfunction. A total of 16 male New Zealand White rabbits were separated equally into four groups: a control group and PBOO groups obstructed for 2, 4 and 8 weeks respectively. Using the RT-PCR and Western blot analysis, a progressive increase of TGF-β1 in penis was found at 2, 4 and 8 weeks after obstruction. Moreover, the biomarkers for oxidative stress or oxidative damage were significantly detected in the penis of rabbits after PBOO, which include the enhancement of 8-hydroxy-2′-deoxyguanosine (8-OHdG) in urine and plasma, plasma malondialdehyde (MDA) and total antioxidant capacity (TAC), as well as reduction of glutathione (GSH). On the basis of our results, the increase of TGF-β1 and elevated systemic oxidative stress may play key roles to contribute to penile dysfunction after chronic PBOO.