Rice is highly sensitive to cold stress, particularly at the booting stage, which significantly threatens rice production. In this study, we cloned a gene, CTB6, encoding a lipid transfer protein involved in cold tolerance at the booting stage in rice, based on our previous fine-mapped quantitative trait locus (QTL) qCTB10-2. CTB6 is mainly expressed in the tapetum and young microspores of the anther. CTB6 interacts with catalases (CATs) to maintain their stability, thereby scavenging reactive oxygen species (ROS) accumulation in anthers and facilitating tapetum development under cold stress conditions. Additionally, CTB6 has lipid-binding ability and affects the lipid content in anthers to regulate cold tolerance at the booting stage. Haplotype analysis and promoter activity assay revealed a specific single nucleotide polymorphism (SNP)-489 variation in the promoter of CTB6, which enhances its expression and results in improved cold tolerance in Hap1-K varieties. The CTB6 near-isogenic line (NIL) exhibited enhanced cold tolerance at the booting stage, with no significant effects on other agronomic traits. Our findings uncover a natural variation of CTB6 for cold tolerance at the booting stage and provide new genetic resources for cold tolerance breeding in rice.