The growth of urban areas represents a leading cause of biodiversity loss and can lead to altered phenotypes. Anthropogenic disturbances might not only cause negative behavioural consequences in animals but also elicit adaptive behavioural responses via plasticity/learning or evolution. One pollutant universally linked with urbanization is artificial light at night (ALAN). ALAN causes widespread biological impacts, but we still know little about its ecological and evolutionary consequences, especially for aquatic organisms. Our field study examined the ecological and phenotypic effects of ALAN in a diurnal, freshwater fish, the Eastern Mosquitofish, Gambusia holbrooki. We observed no evidence for negative effects of ALAN on population demographics or body size. We observed a number of behavioural shifts, some of which matched a priori adaptive hypotheses. While most fish appeared to sleep during the night in populations unexposed to ALAN, we observed a high incidence of night-time activity in ALAN-exposed populations. Active fish swam at a much higher speed at night within ALAN-exposed populations, apparently extending feeding behaviours throughout the night. Based on activity patterns, one population, the one with the longest history and greatest magnitude of ALAN, even displayed a loss of diurnality. Females in ALAN-exposed populations showed reduced daytime feeding rates, perhaps resulting from successful night-time feeding. Meanwhile, males exhibited higher rates of aggression and lower rates of sexual behaviours during the day in populations with greater magnitudes of ALAN. This could reflect proximate consequences of altered circadian rhythms, but could also involve an adaptive shift where males perform more difficult and risky mating behaviours under dim night-time lighting, where they might have higher success and lower risk from predators. For body condition and female daytime behaviours, we found evidence that mixing/gene flow with unexposed populations might have constrained adaptive divergence. Overall, we uncovered how one ubiquitous component of urbanization may have far-reaching consequences that extend beyond immediate, negative biological effects.