Improvement of cold tolerance at the booting stage (CTB) in rice is a key strategy for cultivation in high-altitude and high-latitude regions. Here, we identify CTB3 gene, encoding a calmodulin-binding transcriptional activator that positively regulates cold tolerance at the booting stage in japonica rice. Two indels (57-bp and 284-bp) in the CTB3 promoter confer a differential transcriptional response to cold between the japonica and indica subspecies. OsTCP19 suppresses CTB3 expression by binding to these indels, negatively regulating cold tolerance. CTB3 activates the expression of TREHALOSE-6-PHOSPHATE PHOSPHATASE1 (OsTPP1), reducing trehalose 6-phosphate (Tre6P) levels, which increases sugar accumulation in panicles and improves cold tolerance. Additionally, favorable alleles of OsTCP19 and CTB3 are selected in japonica rice for cold adaptation. These findings highlight the important role of CTB3 in cold adaptation and its potential for improving cold tolerance in rice breeding.