Light pollution is a major anthropogenic environmental change and a significant threat to ecosystems. Among other detrimental effects on physiology, artificial light at night (ALAN) disrupts circadian rhythms in a wide range of species. However, the underlying neuronal and genetic mechanisms remain poorly understood. Here, we show in Drosophila that the loss of the circadian clock gene period exacerbates the ALAN-induced shift towards nocturnal behaviour, with a more pronounced effect on males. In contrast, the loss of cycle has no such effect on males or females; cyc null mutants are nocturnal under standard light‒dark cycles, and their activity and sleep profiles are minimally or not affected by ALAN exposure. CRISPR-Cas9 knockout of period in most clock neurons resembles the null mutant phenotype. Our results show that mutations in components of the positive and negative limbs of the circadian clock result in distinct responses to nocturnal light and highlight the role of genetic factors in modulating behavioural plasticity in response to environmental perturbations.