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The study of nitrogen fixation in sugarcane has a long history that has demonstrated high potential but with substantial variation in results. This 32-month study sought to assess the response of nitrogen fixation associated with sugarcane (Saccharum officinarum L. cvs. ‘Akoki, Honua‘ula, and ‘Ula) to available soil nitrogen. Plants were grown in large pots of perlite along with a fixing and a non-fixing plant control and administered liquid fertigation with varying amounts of isotopically enriched nitrogen. Assessment of nitrogen fixation utilized nitrogen isotope tracing and acetylene reduction assay in the target and control plants. Isotope enrichment and acetylene reduction assay both indicated that nitrogen fixation peaked under low nitrogen application, and declined with higher application rates, with agreement between the two methods. These results suggest that sugarcane engages in a downregulation of nitrogen fixation under high nitrogen availability, potentially explaining the high variation in published experimental results. This suggests that nitrogen management and fertilization strategy can impact the atmospheric inputs of nitrogen in sugarcane cultivation, and the potential to improve nitrogen application efficiency in cropping systems utilizing sugarcane.