Oxalate may cause hypocalcaemia or formation of urinary calculi in animals with prolonged grazing of Napier grass (Pennisetum purpureum) × pearl millet (bajra, P. glaucum) hybrid (NBH). We investigated the influence of nitrate metabolism, nitrogen (N) nutrition, N forms and seasonal variation on oxalate accumulation in leaves of NBH in a field experiment in Ludhiana, India. The experiment was a randomised block design with three N sources (nitrate, amide and ammonium), three application rates (50, 75 and 100 kg N/ha), four seasons (summer, monsoon, autumn, pre-winter) and three replicates. Applied N nutrition induced oxalate synthesis and activities of nitrate reductase (NR) and nitrite reductase (NiR) enzymes. A positive association of N nutrition with both oxalate accumulation and nitrate-reducing enzymes was found. Nitrate-N increased oxalate accumulation and NiR activity more than ammonium and amide. A differential effect of seasons on NR and NiR activities, as well as on oxalate accumulation, was observed. Among different harvest seasons, NR and NiR activities were positively associated with oxalate accumulation in summer and the monsoon season. These results suggest that N fertilisation, particularly in nitrate form, is associated with upregulation of nitrate-reducing enzymes, leading to oxalate accumulation in NBH leaves.