Paeonia ostii is now being extensively planted for oil extraction in China, which is recognized as a single oil-use tree peony cultivar and commonly called 'Fengdan'. This study investigated the effects of nitrogen fertilizer on oil yield, fatty acid compositions and antioxidant activity of P. ostii. Oil yield (33.46 %), oleic acid (25.12 %), linoleic acid (29.21 %) and α-linolenic acid (43.12 %) reached the maximum at N450 treatment, with significant differences compared with other treatments (P<0.05). Furthermore, strong antioxidant activity with low DPPHIC50 value (19.43±1.91 μg mL-1 ) and large ABTS value (1216.53±30.21 μmol Trolox g-1 ) and FRAP value (473.57±9.11 μmol Trolox g-1 ) was also observed at N450. Palmitic acid (5.57 %) and stearic acid (2.02 %) reached a maximum at N375, but not significant with N450 (P<0.05). Nitrogen fertilizer could promote oil yield, fatty acid accumulation and antioxidant activity, and N450 (450 kg ha-1 ) is recommended as the optimum application for P. ostii.