Chilling stress is a major abiotic constraint that affects sweet corn seedling establishment. Spermidine (Spd) is known to participate in the cold stress response in plants, nonetheless, the molecular mechanisms are largely unknown. The present study reported that exogenous Spd not only stimulated seedling growth but also enhanced photosynthetic attributes under chilling stress. Further analysis revealed that Spd application resulted in depressed abscisic acid (ABA) content and affected the expression of ABA-responsive genes. The augmented antioxidant enzymes activities, coupled with declined reactive oxygen species (ROS) content, were observed in response to Spd treatment under chilling stress. Moreover, Spd up-regulated the transcription levels of some Ca2+ transport-related genes. We propose that Spd contributes to cold tolerance in sweet corn seedlings via the regulation of genes involved in the ROS, ABA pathways and Ca2+ transport.