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As the global population urbanizes, dramatic changes are expected in city lighting and the urban form, which may threaten the functioning of urban ecosystems and the services they deliver. However, little is known about the ecological impact of lighting in different urban contexts. Movement is an important ecological process that can be disrupted by artificial lighting. We explored the impact of lighting on gap crossing for Pipistrellus pipistrellus, a species of bat (Chiroptera) common within UK cities. We aimed to determine whether the probability of crossing gaps in tree cover varied with crossing distance and lighting level, through stratified field surveys. We then used the resulting data on barrier thresholds to model the landscape resistance due to lighting across an entire city and explored the potential impact of scenarios for future changes to street lighting. The level of illumination required to create a barrier effect reduced as crossing distance increased. For those gaps where crossing was recorded, bats selected the darker parts of gaps. Heavily built parts of the case study city were associated with large and brightly lit gaps, and spatial models indicate movement would be highly restricted in these areas. Under a scenario for brighter street lighting, the area of accessible land cover was further reduced in heavily built parts of the city. We believe that this is the first study to demonstrate how lighting may create resistance to species movement throughout an entire city. That connectivity in urban areas is being disrupted for a relatively common species raises questions about the impacts on less tolerant groups and the resilience of bat communities in urban centres. However, this mechanistic approach raises the possibility that some ecological function could be restored in these areas through the strategic dimming of lighting and narrowing of gaps.