Mammals use variations in the length of day to anticipate environmental changes and time their reproduction. Light pollution, which affects day length perception, could lead to changes in biological functions. To explore the effects of light pollution on seasonal reproduction, we conducted an experimental study on a strict long-day breeder, the nocturnal gray mouse lemur (Microcebus murinus). Twelve females were randomly exposed to either a moonlight treatment or a light pollution treatment for 5 weeks during the transition from a short- to long-day photoperiod. Daily rhythms of locomotor activity and core temperature were recorded, and the occurrence of estrus and urinary 17-β-estradiol concentrations were monitored. In females exposed to light pollution, the first seasonal estrus occurred while they were still in a short-day photoperiod, 2 weeks earlier than in females exposed to moonlight. Moreover, the daily rhythms profiles of females exposed to light pollution showed significant changes that mimicked long-day acclimatization. This study demonstrates that light pollution can affect daily rhythms and the timing of seasonal estrus in a primate and suggests that functional changes in the circadian system may underlie the adverse effects of light pollution that are observed in wildlife.