Pharmaceutical pollutants are increasingly being detected in ecosystems worldwide. Indeed, approximately 1 in 10 currently manufactured pharmaceuticals have been found in the environment. In this regard, pollution by antidepressants is especially concerning due to their capacity to cause changes to the central nervous system and endocrine system of exposed wildlife, often at disturbingly low concentrations (ng/L). One such pollutant is the antidepressant fluoxetine, which is among the world's most commonly prescribed psychotherapeutics and also one of the most frequently detected in the environment. Alarmingly, the primary target molecule of fluoxetine is evolutionarily conserved across a wide range of non-target species. As a result, fluoxetine can disrupt ecologically significant behavioural and physiological processes in wildlife. Despite this, the effects of environmentally relevant fluoxetine exposure on aquatic biota remain unclear. To address this gap, we investigated the impacts of environmentally realistic fluoxetine exposure-30 and 380 ng/L-on the behaviour and sperm quality of male eastern mosquitofish (Gambusia holbrooki). Specifically, utilising the mosquitofish, we investigated the impacts of fluoxetine on pre-copulatory mating behaviour and post-copulatory sperm traits, as well as, exploratory behaviour and boldness. We focussed on a combination of male behaviours (i.e. reproduction, exploration and boldness) and sperm quality because fluoxetine has the potential to alter the stress response of wildlife, as well as reproduction, through shifts in the hypothalamic-pituitary-adrenal axis. Importantly, changes in these behaviours and sperm traits could have a direct bearing on male fitness. We found that fluoxetine exposure altered the behaviour of fish at field-detected concentrations. Specifically, fluoxetine affected both association time, and copulatory behaviour carried out by males towards females. Interestingly, however, fluoxetine exposure did not impact exploratory behaviour or boldness, with sperm quality measures (i.e. sperm performance and viability) also not being affected. Our results indicate that fluoxetine exposure can impact reproductive behaviour with implications for organismal fitness and community structure.