Constant exposure of tomato fruit to low temperatures (LT) often results in insufficient energy to sustain growth and development. Polyamines (PAs) are essential substances for cell division, development and stress responses in plants. Integrated metabolomics and transcriptome analysis showed that the PA biosynthesis pathway was significantly enriched in Solanum habrochaites (SH). ShODC1 (Ornithine decarboxylase), a key enzyme in the PA synthesis pathway, was highly sensitive to LT stress. Transient silencing of ShODC1 significantly decreased the ion permeability, the antioxidant capacity, and photosynthesis, as well as ShSPDS (Spermidine synthase) and ShSPMS (Spermine synthase) expression and putrescine (Put) accumulation, thus reducing cold tolerance in tomato. This study confirmed that the PA biosynthesis pathway affects the cold tolerance in SH and analyzed the physiological and biochemical functions of ShODC1 under LT stress, providing a new theoretical basis for improving the resistance in cultivated tomato.