Artificial Light at Night (ALAN) is rapidly increasing and so is scientific interest in its ecological and evolutionary consequences. In wild species, ALAN can modify and disrupt biological rhythms. However, experimental proof of such effects of ALAN in the wild is still scarce. Here, we compared diel rhythms of incubation behaviour, inferred from temperature sensors, of female great tits (Parus major) breeding in urban and forest sites. In parallel, we simulated ALAN by mounting LED lights (1.8 lux) inside forest nest boxes, to determine the potentially causal role of ALAN affecting diel patterns of incubation. Urban females had an earlier onset of activity compared to forest females. Experimentally ALAN-exposed forest females were similar to urban females in their advanced onset of activity, compared to unexposed forest birds. Furthermore, forest females exposed to experimental ALAN were more restless at night compared to forest control and urban females. Our findings demonstrate that ALAN can explain the early activity timing in incubating urban great tits, and might also cause sleep disturbance. Consequently, future research needs to address potential effects of ALAN-induced timing on individual fitness and population dynamics, particularly in open-cup nesting species which might be even more exposed to, and affected by, light pollution.