The rapid expansion of artificial light at night (ALAN) across India is increasingly transforming nocturnal environments with significant ecological consequences. This study investigates the rural–urban gradient of light pollution and its ecological implications by analyzing the spatial distribution and intensity of nighttime illumination across selected urban and rural regions of India. Nighttime radiance data derived from the Visible Infrared Imaging Radiometer Suite (VIIRS) for 2024 were integrated with ground-based observations using a Sky Quality Meter (SQM) to evaluate variations in sky brightness and nocturnal light exposure. The analysis focused on major metropolitan centers including Delhi, Mumbai, Hyderabad, Chennai, and Bengaluru, alongside ecologically sensitive rural landscapes such as the Great Himalayan National Park, parts of Odisha, Arunachal Pradesh, and Rajasthan. Spatial analysis was conducted using Geographic Information Systems (GIS), while ecological impacts were assessed through scientific literature review and selected field observations in biodiversity-sensitive environments. The findings reveal a pronounced rural–urban gradient in ALAN intensity, with urban regions exhibiting substantially higher radiance levels that frequently exceed ecological thresholds known to disrupt nocturnal biodiversity. Although rural areas continue to maintain comparatively darker skies, many are increasingly experiencing light intrusion associated with electrification, tourism, infrastructural development, and peri-urban expansion. The study identifies multiple ecological consequences linked to increasing ALAN exposure, including altered behavioral responses in birds and insects, disruption of circadian rhythms, habitat fragmentation, and degradation of nocturnal ecosystem functioning. The results emphasize the urgent need to integrate light pollution assessment and mitigation into India’s environmental governance, urban planning, and biodiversity conservation frameworks. This study contributes to the growing discourse on ALAN as an emerging environmental stressor in rapidly urbanizing regions and highlights the importance of conserving natural darkness for long-term ecological sustainability.