In the context of 17β-estradiol (E2) as an environmental contaminant, this study was designed to test the hypothesis whether it can modulate antioxidant defenses in Dicentrarchus labrax, taking gills as the target organ. Enzymatic (GPX−glutathione peroxidase; CAT-catalase; GR-glutathione reductase; GST-glutathione S-transferase) and non-enzymatic antioxidants (NP-SH-non protein thiols; GSHt-total glutathione) were measured following 10-day exposure to E2 in two different ways: water diluted (WD, 200 or 2,000 ng/L) and intraperitoneally injected (IP, 0.5 or 5 mg/kg). WD exposure caused a single alteration—CAT increase, whereas IP exposure decreased all the enzymatic antioxidants. Similarly, NP-SH and GSHt were reduced by IP exposure. Thus, different E2 exposure routes determined clear differences on the assessed responses. Despite gills close contact with water, their defenses were not strongly affected in WD experiment. Differently, IP injected fish showed an overall decrease in both enzymatic and non-enzymatic antioxidants, more pronounced at the highest concentration, pointing out the E2 oxidative stress inducing potential in fish.