Ecologically relevant indicators of endocrine disruption in fish must be linked with measures of reproductive success. The ability of male fathead minnows (Pimephales promelas) to compete for, maintain, and defend a spawning substrateis paramount to reproductive success. The present study quantified alterations in male fathead minnow reproductive behaviors after exposure to environmentally relevant concentrations (0, 10, 20, or 40 ng/L) of 17α-ethinylestradiol (EE2) for 21 d. A video-based behavioral quantification system examined changes in male-male competitive behaviors (chasing and head-butting) and ability of males to maintain spawning substrates (nibbling and scrubbing). Behaviors analyzed included time under the spawning substrate, frequency of substrate cleaning, and conspecific aggression. Plasma hormone levels (11-ketotestosterone [11-KT], testosterone, and estradiol [E2]), vitellogenin (VTG), secondary male characteristics (tubercle count and dorsal nape pad rank), gonadosomatic index (GSI), and gonad histology also were evaluated. Exposure to 40 ng/L of EE2 decreased the ability of exposed males to compete with control males for spawning substrates (p = 0.09). Furthermore, exposed males displayed reduced frequency of substrate cleaning activities as well as chasing male competitors (p ≤ 0.05). 11-Ketotestosterone, testosterone, and E2 were lower, and VTG was notably higher, in EE2-exposed males compared with control males (p ≤ 0.03). 17α-Ethinylestradiol exposure in males also was associated with reductions in tubercles; lower GSI, gonadal maturity ranks, and number of resorbed tubercles; and presence of an ovipositor (p ≤ 0.001). These data reveal alterations in male reproductive behavior that coincide with decreased hormone levels and secondary sex characteristics. Behavioral endpoints to discern potential ecological consequences in fish exposed to low concentrations of endocrine-disrupting chemicals may provide sensitive and functional indices of effect.