The escalating issue of light pollution in urban environments poses multifaceted challenges, affecting not only the nocturnal sky but also exerting intricate influences on plant physiology. This study delves into the physiological responses of an urban ornamental tree, Populus alba L. clone DI-1, to varying intensities of streetlamp LED night lighting (NL), a shift from traditional High-Pressure Sodium lamps. The investigation “sheds light” on the molecular pathways underlying observed physiological regulations, aiming to offer a comprehensive understanding of the manifold effects of NL on this tree species. NL altered tree architecture, i.e. increased branch length and diameters, underscoring the dynamic response of trees to nocturnal artificial lighting conditions. Regarding tree physiology, the NL-triggered net CO2 assimilation (Pn) during the night resulted in limitations in stomatal conductance during daylight hours. This led to a reduction in Pn, particularly during dawn, hindering the quantum yield for the reduction of end acceptors of PSI. Changes in chlorophyll a-to-b proportion and overall concentration, electron transport chain, and gene expression further highlight the intricate interplay between NL and plant metabolic regulation. Notably, the increased gene expression of sugar transporters in both NL trees suggested a responsive shift in sugar and starch metabolism. This was reflected in the absence of a starch accumulation during daylight hours in NL leaves. The study emphasizes the need for a holistic approach to urban lighting, considering its profound impact on photosynthesizing citizens. These findings highlight the pressing need for the development of innovative lighting spectra with reduced impact on plant physio-chemistry while ensuring visibility for citizen safety.