Over the past five decades, anthropogenic nitrate contamination has increased significantly in many ground and surface water systems, creating the need to understand how nitrate impacts the physiology of aquatic animals. We collected adult male mosquitofish (Gambusia holbrooki) from eight springs in Florida with varying nitrate concentrations (0.2–5.1mg/L NO 3 -N). Fish were evaluated for spring-related differences in body, liver, and gonad size, gonopodium length, muscle testosterone and 11-ketotestosterone (11-KT) concentrations, and sperm counts and viability. Increased nitrate concentration (up to 5mg/L NO 3 -N) was significantly correlated with decreased total sperm counts per spermatozeugmatum, increased adjusted gonopodium length, and increased adjusted testicular weight. Furthermore, we observed that relatively small differences in spring pH (7.0–7.4) were positively associated with muscle 11-KT concentrations. Finally, minor changes in spring water temperature (21.4–22.9°C) was negatively correlated with adjusted testicular and hepatic weights, and positively correlated with total and live sperm counts. We conclude that male mosquitofish reproduction is not the same among the eight springs tested, and that elevated aquatic nitrate concentrations may explain some of the observed variation.