Cold stress severely restricts growth and development, reduces yields, and impairs quality in tomatoes (). Amylase-associated starch degradation and soluble sugar accumulation have been implicated in adaptation and resistance to abiotic stress. Here, we report a β-amylase (BAM) gene, , which plays a central role in tomato cold tolerance. The expression of was triggered by cold stress. knockout using the CRISPR/Cas9 system retarded starch degradation and reduced soluble sugar accumulation in tomato plants, eventually attenuating cold tolerance. Expression analysis revealed that the transcript level was boosted by MeJA. Furthermore, MYC2, an essential component of the JA signaling pathway, could bind to the promoter and directly activate transcription, as revealed by yeast one-hybrid and dual LUC assays. In addition, the suppression of resulted in increased starch accumulation, decreased soluble sugar content, and reduced tolerance to cold stress in tomato plants. Taken together, these findings demonstrate that JA positively regulates β-amylase-associated starch degradation through the MYC2-SlBAM3 module in tomato during cold stress. The results of the present work expand our understanding of the mechanisms underlying gene activation and starch catabolism under cold stress. The regulatory module of can be further utilized to breed tomato cultivars with enhanced cold tolerance.