Urbanization has caused environmental changes, such as urban heat islands, that affect terrestrial ecosystems. However, how and to what extent urbanization affects plant phenology remains relatively unexplored. Here, we investigated the changes in the satellite-derived SOS in 85 large cities across the conterminous United States for the period 2001–2014. We found that (1) the SOS came significantly earlier (6.1 ± 6.3 days) in 74 cities and was significantly weaker (0.03 ± 0.07) in 43 cities when compared with their surrounding rural areas (P < 0.05); (2) the decreased magnitude mainly occurred in cities in relatively cold regions with an annual mean temperature of <17.3°C (e.g., Minnesota, Michigan, and Pennsylvania); and (3) the magnitude of urban-rural difference in both SOS and was primarily correlated with the intensity of UHI. Simulations of two phenology models further suggested that more and faster heat accumulation contributed to the earlier SOS, while a decrease in required chilling led to a decline in magnitude in urban areas. These findings provide the first observational evidence of a reduced covariation between temperature and SOS in major US cities, implying the response of spring phenology to warming conditions in non-urban environments may decline in the future.