Excessive production of reactive oxygen species (ROS) has consistently been described as a cause of erectile dysfunction (ED), though assessments are often made by indirect byproducts in excised tissue. Under conditions of elevated ROS, many tissues will upregulate the antioxidant defense system as a protective mechanism, though this mechanism is understudied in the penis. The objective of this study was to utilize a novel technique to measure in vivo ROS in the penis of rats, and to investigate the expression of antioxidant and anti‐inflammatory genes following 12 weeks of a control diet (CD) or WD. Following dietary intervention (n = 6 CD; n = 6 WD), a microdialysis probe was inserted into the penis of rats under anesthesia. To measure ROS, probes were perfused with saline containing Amplex Ultrared, horseradish peroxidase, and superoxide dismutase (SOD), and fluorescence measured at the outlet. Penile hydrogen peroxide (1.31 vs. 1.96; p = 0.014) and superoxide (1.86 vs. 2.39; p = 0.029) were elevated in response to the WD. The NADPH oxidase subunit p22phox was increased in WD penes (0.36 vs. 0.51; p = 0.038), while a trend of increased p67phox (0.45 vs. 0.63; p = 0.079) was observed in WD. Of the 11 antioxidant genes investigated, only SOD1 was upregulated in WD (0.98 vs. 1.17; p = 0.03), although expression of the anti‐inflammatory gene heme‐oxygenase 1 (HO‐1) was decreased in WD penes (1.74 vs. 0.96; p < 0.01). In conclusion, NADPH oxidase is a probable source of excessive ROS in WD induced ED, while a modest antioxidant defense response may contribute to redox imbalance in the penis. Downregulation of HO‐1 following WD may exacerbate ED through promotion of inflammation.Supported by a fellowship grant from the Sexual Medicine Society of North America.