Urban expansion is a crucial process altering nutrient cycles in agro-ecosystems, often accompanied by negative impacts on the environment. Quantitative analysis of nutrient flows in agricultural systems of megacities and their interactions with urbanization is still lacking. This study reports on changing patterns in inputs, outputs, losses and cycling of nitrogen (N) and phosphorus (P) in agricultural systems in three of China's megacities–Beijing, Shanghai and Chongqing during 1990–2014, using the substance flow analysis method. Results show that changing patterns of nutrient flows varied among cities. With rising urbanization rate in Chongqing, nutrients were increasingly imported to agricultural systems to sustain food demand, which led to increased nutrient losses. An opposite trend occurred in Beijing and Shanghai with high urbanization levels (over 80%) since the early 2000s, resulting from a decline in cropland and livestock numbers, and stricter enforcement of environmental laws. Mineral fertilizers and livestock husbandry both contributed largely to ammonia emissions from agriculture. Losses of nutrients to water bodies in Beijing were sourced mainly from the livestock sector, while derived from overuse of mineral fertilizers in Chongqing, suggesting that priority management practices need to be designed differently among regions. Increased intensity of nutrient losses from agriculture was significantly correlated with increased mineral fertilizer input, livestock density and feed import and with reduced recycling ratio of manure. Integrated management for better use of nutrients in fertilizers, feed and manure are urgently required at regional scales. Our findings can serve as basis for policy decisions for sustainable agricultural systems in megacities.