AbstractFruit softening is one of the main reasons for the loss of fresh blueberry; spermidine (Spd) delays fruit softening to some extent. However, the specific mechanism by which Spd delays fruit softening is not clear. Abscisic acid (ABA) plays a key role in fruit senescence. This study aimed to investigate the effects of exogenous Spd treatment on endogenous ABA accumulation and the softening in postharvest blueberry fruits and identify the key genes in the ABA biosynthesis pathway. Harvested blueberry fruits were immersed in 1.5 mmol L−1 Spd aqueous solution for 10 min. The Spd‐treated fruit showed higher firmness, and lower activities of cell wall metabolism‐related enzymes. Additionally, Spd treatment inhibited the accumulation of endogenous ABA and expression of key genes involved in ABA biosynthesis. The expression of VcNCED3 was significantly negatively correlated with the change in firmness of blueberry fruits. Overexpressing VcNCED3 promotes the accumulation of ABA, thereby exacerbating fruit softening. Therefore, exogenous Spd treatment inhibited the accumulation of ABA by inhibiting the expression of the VcNCED3, thereby delaying the blueberry fruit softening.