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Nitrogen (N) plays an important role in sugarcane (Saccharum spp. hybrids) growth and development; however, long-term effects of N application levels on cane and sugar production in different sugarcane cultivars under field conditions remain unclear. In this study, we investigate the agronomic, yield, and quality traits in three sugarcane cultivars (GT11, B9, and ROC22) under different N levels (0, 150, and 300 kg/ha urea) from 2015 to 2019. Continuous four-year field experiments of plant and ratoon crops were carried out by using two-factor split-plot design. The results showed that N fertilizer application improved the tillering rate, stalk diameter, plant height, stalk weight, millable stalks/ha, cane yield, sugar yield and juice rate of cane, and the difference between N application and non-N application was significant. The cane yield, millable stalks/ha, juice rate, and juice gravity purity increased with the increase of N application, but the milled juice brix and sucrose % cane decreased with the increase of N application. The sugar yield was the highest at 150 kg/ha urea application, while the cane yield was the highest at 300 kg/ha urea application. Different N fertilizer application levels significantly regulated the activities of glutamic pyruvic transaminase (GPT) and glutamic oxaloacetic transaminase (GOT) and the contents of chlorophyll and nitrate N in plant leaves, which reflected the regulation in nitrogen metabolism and alteration in dry matter production and distribution, cane yield and sugar accumulation in different sugarcane cultivars. During the four-year experiment duration, the cane yield and sugar yield generally showed ROC22 > B9 > GT11. These data suggested that 300 kg/ha urea application was suitable for the plant and first ratoon crops, and 150 kg/ha urea application was suitable for the second and third ratoon crops. Both cane and sugar yields could be the highest in a four-year production cycle under this circumstance. [ABSTRACT FROM AUTHOR]