ABSTRACT: N Fertilizer recommendations must be improved to optimize N use efficiency (NUE) for bioenergy crops. A study was conducted to test the hypothesis that sites varying in historical usage of by-product differ in soil N-supplying power and sugarcane (Saccharum spp.) responsiveness to N fertilization. Our aim was to quantify soil N availability and N fertilizer rates, sources, and application timings for their effects on sugarcane yield and NUE. Three N response trials, each involving 0 to 200 kg N ha−1, were conducted in the state of São Paulo, Brazil, at sites varying historically in the usage of vinasse. Before fertilizer application and at harvest, soil inorganic N content was quantified and potential N mineralization estimated by the Illinois Soil Nitrogen Test (ISNT); stalk yield and sugar content were measured at harvest and used to estimate NUE. Sugarcane showed significant response to N fertilization only at the sites with no history of vinasse usage. Reducing the N rate from 120 to 80 kg N ha−1 showed limited potential for lowering yield (~ 1 %), while increasing the NUE by 54 %, which was far better than the 14 % increase achieved by modifying the N source or application timing. Monitoring inorganic N and ISNT levels over time to estimate soil N-supplying power has potential for predicting the responsiveness of sugarcane to N fertilization; however, ISNT interpretations must consider factors that impede mineralization or crop N utilization, such as soil acidity or a limitation on the availability of Ca or P. Soil N testing can help optimize NUE for sustainable bioenergy production.