Phthalate ester plasticizers are antiandrogenic in mammals. High doses of certain phthalates consistently interfere with the normal development of male offspring exposed in utero, causing disrupted sperm production, abnormal development of the genitalia, and in some cases infertility. In the environment, phthalates are considered ubiquitous and are commonly measured in aquatic ecosystems at low nanograms to micrograms per liter concentrations. Given the similarity between mammalian and teleost endocrine systems, phthalate esters may be able to cause antiandrogenic endocrine disruption in fish in the wild. In the present study, adult male three-spined sticklebacks (Gasterosteus aculetaus; n = 8) were exposed to di-n-butyl phthalate (DBP; 0, 15, and 35 µg DBP/L) for 22 d and analyzed for changes in nesting behavior, plasma androgen concentrations, spiggin concentrations, and steroidogenic gene expression. Plasma testosterone concentrations were significantly higher in males from the 35 µg DBP/L group compared with the solvent control, whereas plasma 11-ketotestosterone concentrations were not significantly affected. Expression of steroid acute regulatory protein and 3β-hydroxysteroid dehydrogenase remained unchanged. Spiggin concentrations were significantly lower in the males exposed to 35 µg DBP/L. Nest building appeared to be slower in some males exposed to DBP, but this was not statistically significant. These results suggest that DBP has antiandrogenic effects in fish. However, further research is required to firmly establish the consequences of chronic DBP exposure in fish.